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ESSENTIALS  OF 
BACTERIOLOGY 

M.V.BALL,  M.D. 


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Fifth  Edition,  Just  Ready  "With  Complete  Vocabulary 

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MEDICAL  DICTIONARY 

•  EDITED  BY 

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Assistant   Demonstrator  of   Obstetrics,   University   of   Pennsylvania. 

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lary. Besides  the  ordinary  dictionary  terms  the  book  contains 
a  wealth  of  anatomical  and  other  tables.  This  matter  is 
of  particular  value  to  students  for  memorizing  in  preparation 
for  examination. 

"  I  am  struck  at  once  with  admiration  at  the  compact  size  and  attractive  ex- 
terior. I  can  recommend  it  to  our  students  without  reserve." — JAMES  W.  HOL- 
LAND, M.  D.,  of  Jefferson  Medical  College. 

"  This  is  a  handy  pocket  dictionary,  which  is  so  full  and  complete  that  it  puts 
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W.  B.  SAUND^RS  COMPANY,  925  Walnut  St.,  Phila. 

London:  9,  Henrietta  Street,  Covent  Garden 


ESSENTIALS 


OF 


BACTERIOLOGY 


SINCE  the  issue  of  the  first  volume  of  the 
Saunders  Question=Compends, 

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Objectives  and 
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BACTERIOLOGIC  MICROSCOPE. 


SAUNDERS'   QUESTION-COMPENDS,    No.  20 


ESSENTIALS 


OF 


BACTERIOLOGY 


BEING  A 


CONCISE  AND  SYSTEMATIC  INTRODUCTION  TO  THE 
STUDY  OF  BACTERIA  AND  ALLIED  MICROORGANISMS 


BY 

M.   V.   BALL,   M.D. 

MEMBER    OF    THE     ACADEMY    OF    NATURAL    SCIENCES    OF     PHILADELPHIA;     CONSULTING 
OPHTHALMOLOGIST  AND  JURIST  TO  THE  STATE  HOSPITAL  AT  WARREN,  PA.; 
WARREN  COUNTY    MEDICAL  INSPECTOR  FOR     PENNSYLVANIA,  DE- 
PARTMENT  OF   HEALTH;   FORMERLY  INSTRUCTOR   IN  BAC- 
TERIOLOGY   AT    THE   PHILADELPHIA  POLYCLINIC. 


SIXTH    EDITION,   THOROUGHLY    REVISED 


With  135  Illustrations,  some  in  Colors 


PHILADELPHIA   AND   LONDON 

B,    SAUNDERS    COMPANY 

J908 


Main  Lib. 
Agric,  Dept. 


LIBRARY  Of  CONGRESS 
Two  Copies  Received 

OCT    23  1908 

Copyright  Entry 


BIOLOGY 

LIBRARY 

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Set  up,  electrotyped,  printed,  and  copyrighted  October,  1891.      Reprinted  October, 
1892.     Revised,  reprinted,  and  recopyrighted  May,  1893.     Reprinted  June,  1894. 
Revised,  reprinted,  and  recopyrighted  November,  1896.     Reprinted  Octo- 
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printed May,  1903.     Revised,  reprinted,  and  recopyrighted 
August,  1904.     Reprinted  October,  1905,  and  August, 
1907.     Revised,  entirely  reset,  reprinted,  and 
recopyrighted  September,  1908. 


Copyright,  1908,  by  W.  B.  Saunders  Company. 


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PRINTED    IN    AMERICA 


PRESS    OF 

W.    B.    SAUNDERS   COMPANY 
PHILADELPHIA 


PREFACE  TO  THE  SIXTH  EDITION 


THIS  edition  has  been  thoroughly  revised  and  new  chapters 
on  Protozoa  and  Opsonic  Technic  added.  While  this  work  is 
intended  as  an  introduction  only,  I  have  endeavored  to  make 
it  complete  enough  to  permit  the  student  and  the  practitioner 
by  its  aid  to  examine  and  identify  any  or  all  of  the  important 
bacteria.  It  aims  to  contain  the  essentials  of  larger  works  and 
at  the  same  time  serve  as  a  guide  in  the  laboratory  of  the 
student,  the  clinician,  and  the  sanatarian. 

Medical  bacteriology  has  gradually  broadened  its  scope,  so 
that  now  the  accomplished  bacteriologist  must  be  well  versed 
not  only  in  a  knowledge  of  allied  microorganisms,  but  also  in 
the  knowledge  of  the  higher  life  forms,  the  normal  and  path- 
ologic anatomy  of  birds,  mosquitoes,  flies,  rats,  and  fleas, 
through  whom  important  diseases  are  transmitted  to  man. 
Although  from  the  beginning  bacteriology  has  been  closely 
associated  with  zoology,  never  before  -has  the  subject  required 
such  an  extensive  knowledge  of  ..all  biologic  sciences,  to  say 
nothing  of  the  depths  in  chemistry  the  studies  of  immunity 
have  gradually  entered.  Elementary  microbiology  should  be 
taught  in  close  association  with  other  sciences  and,  therefore, 
in  a  university  as  a  preparatory  course,  while  an  advanced 
course  only  should  be  taught  in  medical  schools  in  connection 
with  pathology. 

M.  V.  BALL. 

WARREN,  PA.,  October,  1908. 

7 

268469 


PREFACE  TO   THE  FIRST   EDITION 


FEELING  the  need  of  a  Compendium  on  the  subject  of 
this  work,  it  has  been  our  aim  to  produce  a  concise  treatise 
upon  the  Practical  Bacteriology  of  to-day,  chiefly  for  the 
medical  student,  which  he  may  use  in  his  laboratory. 

It  is  the  result  of  experience  gained  in  the  Laboratory 
of  the  Hygienical  Institute,  Berlin,  under  the  guidance  of 
Koch  and  Frankel;  and  of  information  gathered  from  the 
original  works  of  other  German,  as  well  as  of  French,  bac- 
teriologists. 

Theory  and  obsolete  methods  have  been  slightly  touched 
upon.  The  scope  of  the  work  and  want  of  space  forbade 
adequate  consideration  of  them.  The  exact  measurements 
of  bacteria  have  not  been  given.  The  same  bacterium  varies 
often  much  in  size,  owing  to  differences  in  the  media,  staining, 
etc. 

We  have  received  special  help  from  the  following  books, 
which  we  recommend  to  students  for  further  reference: 

MACE:  Traite  pratique  de  Bacteriologie. 
FRANKEL:  Grundriss  der  Bakterienkunde. 
EISENBERG:  Bakteriologische  Diagnostik. 
CROOKSHANK,  E.  M.:  Manual  of  Bacteriology. 
GUNTHER:  Einfiihring  in  das  Studium  der  Bacteriologie,  etc. 
WOODHEAD  and  HARE:  Pathological  Mycology. 
SALMONSEN:  Bacteriological    Technique    (English    transla- 
tion). 

M.  V.  BALL. 
8 


CONTENTS 


PART  I 
GENERAL  CONSIDERATIONS   AND   TECHNIC 

PAGE 

INTRODUCTION 15 

CHAPTER  I. — CLASSIFICATION,   STRUCTURE,  AND   REPRO- 
DUCTION    17 

"        II. — ORIGIN,  LIFE,  GROWTH,  AND  PROPERTIES..   22 

"      III. — METHODS  OF  EXAMINATION 26 

"      IV. — STAINING  OF  BACTERIA 30 

"        V. — GENERAL  METHOD  OF  STAINING  SPECIMENS..  36 

"      VI. — SPECIAL  METHODS  OF  STAINING 40 

"     VII. — METHODS  OF  CULTURE 44 

"  VIII. — NUTRIENT  MEDIA 49 

"      IX. — SOLID  TRANSPARENT  MEDIA 53 

"        X. — INOCULATION  OF  GELATIN  AND  AGAR 62 

"      XL — GROWTH  AND  APPEARANCES  OF  COLONIES..   68 

"    XII. — CULTIVATION  OF  ANAEROBIC  BACTERIA 71 

"  XIII.— INFECTION 75 

"   XIV. — IMMUNITY 78 

"     XV. — ANIMAL  EXPERIMENTS 84 

"   XVL— OPSONIC  TECHNIC.  .  87 


PART  II 

SPECIAL  BACTERIOLOGY 

CHAPTER  XVII. — NON-PATHOGENIC  BACTERIA 91 

Bacillus  Prodigiosus 91 

Indicus 92 

9 


IO  CONTENTS 

PAGE 

Bacillus  Mesentericus  Vulgatus 92 

Megaterium 93 

Ramosus 93 

Bacterium  Zopfi 94 

Bacillus  Subtilis 94 

Spinosus 95 

Some  Bacteria  in  Milk 95 

Bacillus  Acidi  Lactici 95 

Boas-Oppler 96 

Butyricus 96 

Amylobacter 97 

Lactis  Cyanogenus 97 

Erythrogenes 98 

Examination  of  Milk  in  Stained  Specimens 98 

Some  Non-Pathogenic  Bacteria  of  Water 99 

Bacillus  Violaceus 99 

Cceruleus. 99 

Fluorescent  Bacteria 99 

Phosphorescent  Bacteria 100 

Leptothrix,  Crenothrix,  Cladothrix,  and 

Beggiatoa 101 

Microorganisms  found  in  Urine 102 

Spirillum 103 

Rubrum;  Concentricum 103 

Sarcina 104 

Lutea 104 

Aurantica  Flava,  Rosea,  and  Alba 105 

Ventriculi 105 

CHAPTER  XVIII. — PATHOGENIC  BACTERIA 105 

Bacteria  Pathogenic  for  Man  and  Other  Animals 105 

Bacillus  Anthracis 105 

Tuberculosis 109 

Lepra  Bacillus 1 20 


CONTENTS  II 

PAOK 

Syphilis  Bacillus 120 

Bacillus  of  Glanders 121 

Mallein 123 

Bacillus  of  Diphtheria 123 

of  Typhoid  Fever 129 

Paracolon  or  Paratyphoid. 136 

Psittacosis 137 

Coli  Communis 137 

CHAPTER  XIX. — PATHOGENIC  BACTERIA — CONTINUED..  .  140 

Spirillum  Choleras 140 

Bacteria  Similar  to  Spirillum  Choleras 146 

Finkler-Prior 146 

Tyrogenum 147 

Vibrio  Metschnikovi 147 

Bacteria  of  Pneumonia 148 

Pneumobacillus  of  Friedlander 150 

of  Frankel 151 

Antitoxin  of  Pneumonia 153 

Bacillus  of  Rhinoscleroma 153 

Diplococcus  Intracellularis  Meningitidis 153 

Micrococcus  Tetragenus 155 

Capsule  Bacillus 156 

Bacillus  of  Influenza 157 

Microorganisms  of  Suppuration 158 

Streptococcus  Pyogenes 159 

Staphylococcus  Pyogenes  Aureus 160 

Pyogenes  Albus 162 

Micrococcus  Pyogenes  Citreus 162 

Cereus  Albus 162 

Flavus 162 

Pyogenes  Tenuis 162 

Bacillus  Pyocyaneus 163 

Koch-Weeks'  Bacillus 165 


12  CONTENTS 

PAGE 

Morax-Axenfeld  Diplobacillus  of  Conjunctivitis.  .  166 

Micrococcus  Gonorrhceae 166 

Microbes  Similar  to  Gonorrhea 168 

Protective  Vaccines  in  Pus  Infections 170 

Bacillus  of  Tetanus 170 

(Edematis  Maligni 174 

of  Soft  Chancre 177 

of  Bubonic  Plague 177 

of  Dysentery 179 

Aerogenes  Capsulatus 180 

Micrococcus  Melitensis - 181 

Bacillus  Enteritidis  Sporogenes 182 

Protozoa 183 

Life  Cycle  of  the  Malarial  Sporozoa 184 

Three  Forms  of  Malarial  Protozoa 187 

Methods  of  Examination  for  Malarial  Organ- 
isms   189 

Trypanosoma 1 90 

Lewisi 191 

Brucei 191 

Ugandense  Gambiense 192 

Evansi 192 

Piroplasma  Bovis 192 

Negri  Bodies 193 

Spirillum  of  Relapsing  Fever 193 

Spirochaete  Pallida 194 

Amceba  Dysenteriae 195 

Small-pox  and  Vaccinia 196 

Yellow  Fever 196 

CHAPTER  XX. — BACTERIA  PATHOGENIC  FOR  ANIMALS,  BUT 

NOT  FOR  MAN 196 

Bacillus  of  Symptomatic  Anthrax 196 

of  Chicken  Cholera..  .  198 


CONTENTS  13 

PAGE 

Bacteria  of  Hemorrhagic  Septicemia,  Swine  Plague, 

Duck  Cholera,  etc '. 200 

Bacillus  of  Erysipelas  of  Swine 201 

Murisepticus 202 

Micrococcus  of  Mai  de  Pis 203 

Bacillus  Alvei 204 

Bacterium  Termo 205 

Proteus  Vulgaris 205 

Mirabilis 205 

Zenkeri 205 

CHAPTER  XXL — YEASTS  AND  MOULDS 206 

Oi'diums 207 

Cladothrices  and  Streptothrices 212 

Streptothrix     or     Cladothrix     Actinomyces 

(Ray-fungus) 212 

Madurae 214 

Farcinica 215 

CHAPTER  XXII. — EXAMINATION    OF    AIR,    SOIL,    AND 

WATER 216 

The  Bacteria  of  Milk  and  Other  Foods 228 

CHAPTER  XXIII. — BACTERIOLOGIC  EXAMINATION  OF  THE 

ORGANS  AND  CAVITIES  OF  THE  HUMAN  BODY.  .  229 

CHAPTER  XXIV.— ANTISEPTICS  AND  ANTISEPSIS 233 

Tables    of    Chief    Characteristics    of    the  Principal 

Bacteria 238 

Part  I.  Non-Pathogenic *. . .  238 

Part  II.  Pathogenic 262 


ESSENTIALS  OF  BACTERIOLOGY 


INTRODUCTION 


History.— The  microscope  was  invented  about  the  latter 
part  of  the  sixteenth  century,  and  soon  after,  by  its  aid,  minute 
organisms  were  found  in  decomposing  substances.  Kircher, 
in  1646,  suggested  that  diseases  might  be  due  to  similar  organ- 
isms, but  the  means  at  his  disposal  were  insufficient  to  enable 
him  to  prove  his  theories.  Anthony  Van  Leeuwenhoek,  of 
Delft,  Holland  (1680  to  1723),  so  improved  the  instrument 
that  he  was  enabled  thereby  to  discover  microorganisms  in 
vegetable  infusion,  saliva,  fecal  matter,  and  scrapings  from 
the  teeth.  He  distinguished  several  varieties,  showed  them 
to  have  the  power  of  locomotion,  and  compared  them  in  size 
with  various  grains  of  definite  measurement.  It  was  a  great 
service  that  this  "Dutch  naturalist"  rendered  the  world;  and 
he  can  rightly  be  called  the  " father  of  microscopy." 

Various  theories  were  then  formulated  by  physicians  to 
connect  the  origin  of  different  diseases  with  bacteria;  but  no 
proofs  of  the  connection  could  be  obtained.  Andry,  in  1701, 
called  bacteria  worms.  Miiller,  of  Copenhagen,  in  1786,  made 
a  classification  composed  of  two  main  divisions — monas  and 
vibrio;  and  with  the  aid  of  the  compound  microscope  was 
better  able  to  describe  them.  Ehrenberg,  in  1833,  with  still 
better  instruments,  divided  bacteria  into  four  orders:  bac- 
terium, vibrio,  spirillum,  and  spirochaete.  It  was  not  until 


1 6  ESSENTIALS    OF    BACTERIOLOGY 

i8t>3  that  any  positive  advance  was  made  in  connecting  bacteria 
with  disease.  Rayer  and  Davaine  had,  in  1850,  already  found 
a  rod-shaped  bacterium  in  the  blood  of  animals  suffering 
from  splenic  fever  (sang  de  rate),  but  they  attached  no  special 
significance  to  their  discovery  until  Pasteur  made  public  his 
grand  researches  in  regard  to  fermentation  and  the  role  bacteria 
played  in  the  economy.  Then  Davaine  resumed  his  studies, 
and  in  1863  established  by  experiments  the  bacterial  nature 
of  splenic  fever  or  anthrax. 

But  the  first  complete  study  of  a  contagious  affection  was 
made  by  Pasteur  in  1869,  in  the  diseases  affecting  silk- worms, 
— pebrine  and  flacherie, — which  he  showed  to  be  due  to  micro- 
organisms. 

Then  Koch,  in  1875,  described  more  fully  the  anthrax  bacillus, 
gave  a  description  of  its  spores  and  the  properties  of  the  same, 
and  was  enabled  to  cultivate  the  germ  on  artificial  media; 
and,  to  complete  the  chain  of  evidence,  Pasteur  and  his  pupils 
supplied  the  last  link  by  reproducing  the  same  disease  in  animals 
by  artificial  inoculation  from  pure  cultures.  The  study  of  the 
bacterial  nature  of  anthrax  has  been  the  basis  of  our  knowledge 
of  all  contagious  maladies,  and  most  advances  have  been  made 
first  with  the  bacterium  of  that  disease. 

Since  then  bacteriology  has  grown  to  huge  proportions — 
become  a  science  in  itself — and  thousands  of  earnest  workers 
are  adding  yearly  solid  blocks  of  fact  to  the  structure,  which 
structure  it  will  be  our  aim  briefly  to  describe  in  the  pages 
which  are  to  follow. 


BACTERIA 

PART   I 
GENERAL  CONSIDERATIONS 


CHAPTER  I 

.. 

BACTERIA 

THE  bacteria  occupy  the  lowest  plane  of  plant  life  known  to 
us,  though  they  are  by  no  means  as  primitive  in  their  biology 
as  was  formerly  supposed,  and  it  is  quite  possible  that  still 
simpler  forms  may  be  discovered. 

The  numerous  unicellular  vegetable  organisms  which  form 
the  lower  limit  of  plant  life  as  we  know  it  multiply  by  fission 
and  are  hence  called  the  Schizophyta,  or  splitting  plants.  This 
group  is  subdivided  into  two  classes — (a)  the  Schizophycece,  or 


-rX 


a  b  c 

Fig.  i. — Types  of  bacteria:  a,  Micrococcus;  b,  spirillum;  c,  bacillus. 

fission  algae,  and  (6)  the  Schizomycetes,  or  fission  fungi,  or 
bacteria,  as  we  usually  call  them. 

Bacteria  are  unicellular  masses  of  protoplasm  of  micro- 
scopic size,  multiplying  by  fission  and  existing  without  chloro- 
phyl.  Three  main  types  are  found:  (i)  Globular  forms, 
called  cocci;  (2)  straight  rod-shaped  forms,  called  bacilli;  (3) 
curved  or  spiral  rods,  called  spirilla.  (See  Fig.  i.) 

Structure. — Bacteria  are  cells;  they  appear  as  round  or 
cylindric,  of  an  average  diameter  on  transverse  section  of  o.ooi 


1 8  ESSENTIALS    OF    BACTERIOLOGY 

mm.  (=i  micromillimeter),  written  i  //.  The  cell,  as  other 
plant-cells,  is  composed  of  a  membranous  cell-wall  and  cell- 
contents;  nuclei  are  not  found. 

Cell-wall. — The  cell-wall  is  composed  either  of  plant  cellu- 
lose, or  a  form  of  albumin,  since  it  is  less  permeable  than  cellu- 
lose membrane.  The  membrane  is  firm,  and  can  be  brought 
plainly  into  view  by  the  action  of  iodin  upon  the  cell-contents, 
which  contract  tnem. 

Cell-contents. — The  contents  of  the  cell  consist  mainly  of 
protoplasm,  usually  homogeneous,  but  in  some  varieties  finely 


Fig.  2. — Zobglea. 

granular,  or  holding  pigment,  chlorophyl,  fat-droplets,  and  sul- 
phur in  its  structure.  It  permits  osmosis,  -and  is  like  other 
plant-cells. 

Gelatinous  Membrane. — The  outer  layer  of  the  cell-mem- 
brane can  absorb  water  and  become  gelatinoid,  forming  either 
a  little  envelop  or  capsule  around  the  bacterium  or  preventing 
the  separation  of  the  newly  branched  germs,  forming  chains  and 
bunches,  as  streptococci  and  staphylococci.  Long  filaments 
are  also  formed. 

Zobglea. — When  this  gelatinous  membrane  is  very  thick, 
irregular  masses  of  bacteria  will  be  formed,  the  whole  growth 


BACTERIA  19 

being  in  one  jelly-like  lump.  This  is  termed  a  zooglea  (C£>ov, 
animal,  /A«««9,  glue)  (Fig.  2). 

Locomotion. — Many  bacteria  possess  the  faculty  of  self- 
movement,  carrying  themselves  in  all  manner  of  ways  across 
the  microscopic  field — some  very  quickly,  others  leisurely. 

Vibratory  Movements. — Some  bacteria  vibrate  in  them- 
selves, appearing  to  move,  but  they  do  not  change  their  place; 
these  movements  are  denoted  as  molecular  or  "Brownian," 
and  are  due  to  purely  physical  causes. 

Flagella. — Little  threads  or  lashes  are  found  attached  to 
many  of  the  motile  bacteria,  either  at  the  poles  or  along  the 


a  b  c 

Fig.  3. — Types  of  flagella:  a,  Vibrio  cholerae,  one  flagellum  at  the  end — 
monotrichia  type;  b,  Bacterium  syncyaneum,  tuft  of  flagella  at  the  end,  rarely 
at  the  side — lophotrichia  type;  c,  Bacterium  vulgare,  flagella  arranged  all 
about — peritrichia  type  (Lehmann  and  Neumann). 

sides — sometimes  only  one,  and  on  some  several,  forming  a 
tuft. 

These  flagella  are  m  constant  motion,  and  can  probably  be 
considered  as  the  organs  of  locomotion;  they  have  not  been 
discovered  upon  all  the  motile  bacteria,  owing,  no  doubt,  to  our 
imperfect  methods  of  observation.  They  can  be  stained  and 
have  been  photographed.  (See  Fig.  3.)  Flagella  serve  some- 
times to  increase  food-supply,  and  have  been  found  on  some 
species  which  are  non-motile. 

Reproduction. — Bacteria  multiply  through  simple  division 
or  fission,  as  it  is  called.  Spore  formation  is  simply  a  resting 
stage  and  not  a  means  of  multiplication.  To  accomplish 
division  the  cell  elongates,  and  at  one  portion,  usually  the 
middle,  the  cell-wall  indents  itself  gradually,  forming  a  septum 


20  ESSENTIALS    OF    BACTERIOLOGY 

and  dividing  the  cell  into  two  equal  parts,  just  as  occurs  in  the 
higher  plant  and  animal  cells.  (See  Fig.  4.) 

Successive  divisions  take  place,  the  new  members  either  exist- 
ing as  separate  cells  or  forming  part  of  a  community  or  group. 
It  has  been  computed  that  if  division  takes  place  every  hour, 
as  it  often  does,  one  individual  in  twenty-four  hours  will  have 
17,000,000  descendants. 

Spore  Formations. — Two  forms  of  sporulation,  endosporous 
and  arthrosporous. 

Endosporous. — First,  a  small  granule  develops  in  the  pro- 
toplasm of  a  bacterium;  this  increases  in  size,  or  several  little 


Fig.  4. — Division  of  bacteria:   a,  Division  of  a  micrococcus;  b,  division  of 
a  bacillus  (after  Mace). 

granules  coalesce  to  form  an  elongated,  highly  refractive, 
clearly  denned  object,  rapidly  attaining  its  real  size,  and  this 
is  the  spore.  The  remainder  of  the  cell-contents  has  now 
disappeared,  leaving  the  spore  in  a  dark,  very  resistant  mem- 
brane or  capsule,  and  beyond  this  the  weak  cell-wall.  The 
cell-wall  dissolves  gradually  or  stretches  and  allows  the  spore 
to  be  set  free. 

Each  bacterium  gives  rise  to  but  one  spore.  It  may  be  at 
either  end  or  in  the  middle  (Fig.  5) .  Some  rods  take  on  a  pecu- 
liar shape  at  the  site  of  the  spore,  making  the  rod  look  like  a 
drum-stick  or  spindle — clostridium  (Fig.  6). 

Spore  Contents.— What  the  real  contents  of  spores  are  is  not 


BACTERIA 


21 


known.  In  the  mother-cell  at  the  site  of  the  spore  little  gran- 
ules have  been  found  which  stain  differently  from  the  rest  of 
the  cell,  and  these  are  supposed  to  be  the  beginnings — the  sporo- 
genic  bodies.  The  most  important  part  of  the  spore  is  its  cap- 
sule; to  this  it  owes  its  resisting  properties.  It  consists  of  two 
separate  layers — a  thin  membrane  around  the  cell,  and  a  firm 
outer  gelatinous  envelop. 

Germination. — When  brought  into  favorable  conditions,  the 
spore  begins  to  lose  its  shining  appearance,  the  outer  firm  mem- 
brane begins  to  swell,  and  it  now  assumes  the  shape  and  size 


Fig.  5. — Sporulation  (after  De  Bary). 


Fig.  6. — Clostridium. 


of  the  cell  from  which  it  sprang,  the  capsule  having  burst, 
so  as  to  allow  the  young  bacillus  to  be  set  free. 

Requisites  for  Spore  Formation. — It  was  formerly  thought 
that  when  the  substratum  could  no  longer  maintain  it,  or  had 
become  infiltrated  with  detrimental  products,  the  bacterium- 
cell  produced  spores,  or  rather  turned  itself  into  a  spore  to 
escape  annihilation;  but  we  believe  now  that  only  when  con- 
ditions are  the  most  favorable  to  the  well-being  of  the  cell, 
does  it  produce  fruit,  just  as  with  every  other  type  of  plant  or 
animal  life,  a  certain  amount  of  oxygen  and  heat  being  neces- 
sary for  good  spore  formation.  The  question  is  still  unsettled, 
however. 


ESSENTIALS    OF    BACTERIOLOGY 

Asporogenic  Bacteria. — Bacteria  can  be  so  damaged  that 
they  will  remain  sterile — not  produce  any  spores.  This  con- 
dition can  be  temporary  only  or  permanent. 

Arthrosporotrs. — In  the  other  group,  called  arthrospores, 
individual  members  of  a  colony  or  aggregation  leave  the  same, 
and  become  the  originators  of  new  colonies,  thus  assuming  the 
character  of  spores. 

The  micrococci  furnish  examples  of  this  form. 

Some  authorities  have  denied  the  existence  of  the  arthro- 
sporous  formation. 

Resistance  of  Spores. — Because  of  the  very  tenacious 
envelop,  the  spore  is  not  easily  influenced  by  external  measures. 
It  is  said  to  be  the  most  resisting  object  of  the  organic  world. 

Chemical  and  physical  agents  that  easily  destroy  other  life 
have  very  little  effect  upon  it. 

Many  spores  require  a  temperature  of  140°  C.  dry  heat  for 
several  hours  to  destroy  them.  The  spores  of  a  variety  of 
potato  bacillus  (Bacillus  mesentericus)  can  withstand  the 
application  of  steam  at  100°  C.  for  four  hours. 


CHAPTER  II 
ORIGIN     OF    BACTERIA    AND     THEIR     DISTRIBUTION 

As  Pasteur  has  shown,  all  bacteria  develop  from  preexisting 
bacteria  or  the  spores  of  the  same.  They  cannot  arise  out  of 
nothing. 

The  wide  and  almost  universal  diffusion  of  bacteria  is  due  to 
the  minuteness  of  the  cells  and  the  few  requirements  for  their 
existence.  In  a  drop  of  water  1,700,000,000  cocci  can  find 
space. 

Very  few  places  are  free  from  germs;  the  air  on  the  high  seas 
and  on  the  mountain-tops  is  said  to  be  free  from  bacteria, 
but  this  is  questionable. 


ORIGIN    OF   BACTERIA   AND   THEIR   DISTRIBUTION       23 

One  kind  of  bacterium  will  not  produce  another  kind.  A 
bacillus  does  not  arise  from  a  micrococcus,  or  the  typhoid  fever 
bacillus  produce  the  bacillus  of  tetanus. 

This  subject  has  been  long  and  well  discussed,  and  it  would 
take  many  pages  to  state  the  "pros"  and  "cons";  therefore  this 
positive  statement  is  made,  it  being  the  position  now  held  by 
the  principal  authorities. 

Saprophytes  and  Parasites  (Saprophytes:  <ra^o?,  putrid; 
<f>!>Toy,  plant.  Parasites:  xapd,  aside  of;  <T~ITOS,  food). — 
Those  bacteria  which  live  on  the  dead  remains  of  organic  life 
are  known  as  saprophytic  bacteria,  and  those  which  choose  the 
living  bodies  of  their  fellow-creatures  for  their  habitat  are  called 
parasitic  bacteria.  Some,  however,  develop  equally  well  as 
saprophytes  and  parasites.  They  are  called  facultative  para- 
sites. 

Conditions  of  Life  and  Growth  of  Bacteria.— Influence 
of  Temperature. — In  general,  a  temperature  ranging  from  10° 
C.  to  40°  C.  is  necessary  to  their  life  and  growth. 

Saprophytes  take  the  lower  temperatures;  parasites,  the  tem- 
perature more  nearly  approaching  the  animal  heat  of  the  warm 
blooded.  Some  forms  require  a  nearly  constant  heat,  growing 
within  very  small  limits,  as  the  bacillus  of  tuberculosis. 

Some  forms  can  be  arrested  in  their  development  by  a  warmer 
or  colder  temperature,  and  then  restored  to  activity  by  a  return 
to  the  natural  heat. 

A  few  varieties  exist  only  at  freezing-point  of  water,  and 
others  again  will  not  live  under  a  temperature  of  60°  C. 

For  the  majority  of  bacteria  a  temperature  of  60°  C.  is 
destructive;  and  several  times  freezing  and  thawing  very  fatal. 

Influence  of  Oxygen. — Two  varieties  of  bacteria  in  relation 
to  oxygen — the  one  aerobic,  growing  in  air;  the  other,  anae- 
robic, living  without  air. 

Obligate  aerobins,  those  which  exist  only  when  oxygen  is 
present. 

Facultative  aerobins,  those  that  live  best  when  oxygen  is 
present,  but  can  live  without  it. 

Obligate  or  true  anaerobins,  those  which  cannot  exist  where 


24          ESSENTIALS  OF  BACTERIOLOGY 

oxygen  is;  facultative  anaerobins,  those  which  exist  better 
where  there  is  no  oxygen,  but  can  live  in  its  presence. 

Some  derive  the  oxygen  which  they  require  out  of  their  nutri- 
ment, so  that  a  bacterium  may  be  aerobic  and  yet  not  require 
the  presence  of  free  oxygen. 

Aerobins  may  consume  the  free  oxygen  of  a  region  and  thus 
allow  the  anaerobins  to  develop.  By  improved  methods  of 
culture  many  varieties  of  anaerobins  have  been  discovered. 

Influence  of  Light. — Sunlight  is  very  destructive  to  bacteria. 
A  few  hours'  exposure  to  the  sun  has  been  fatal  to  anthrax 
bacilli  and  the  cultures  of  Bacillus  tuberculosis.  The  sun's 
rays,  however,  must  come  in  direct  contact  with  the  germs,  and 
are  usually  active  only  on  the  surface  cultures.  The  rays  at 
the  violet  end  of  the  spectrum  are  the  most  active.  The  electric 
arc-light  has  much  the  same  effect  as  sunlight  on  bacteria. 

Effects  of  Electricity. — Electricity  arrests  growth. 

Effects  ofRontgenRays. — Have  little  or  no  effect  on  artificial 
cultures,  but  in  the  living  tissues  a  pronounced  bactericidal 
effect  is  produced,  perhaps  through  the  stimulation  of  the 
body-cells. 

Vital  Actions  of  Microbes.— Bacteria  feeding  upon  organic 
compounds  produce  chemical  changes  in  them,  not  only  by  the 
withdrawal  of  certain  elements,  but  also  by  the  excretion  of 
these  elements  changed  by  digestion.  Sometimes  such  changes 
are  destructive  to  themselves,  as  when  lactic  and  butyric  acids 
are  formed  in  the  media. 

Oxidation  and  reduction  are  carried  on  by  some  bacteria. 
Ammonia,  hydrogen  sulphid,  and  trimethylamin  are  a  few  of 
the  chemical  products  produced  by  bacteria.  Nitrites  in  the 
soil  are  reduced  to  ammonia. 

Nitrification. — Albuminoids  changed  into  indol,  skatol, 
leucin,  etc.;  then  these  into  ammonia,  ammonia  into  nitrites, 
nitrites  into  nitrates. 

Ptomains. — Brieger  found  a  number  of  complex  alkaloids 
closely  resembling  those  found  in  ordinary  plants,  and  which 
he  named  ptomains,  from  xrwfjLa,  corpse,  because  obtained 
from  putrefying  objects. 


ORIGIN   OF    BACTERIA   AND   THEIR    DISTRIBUTION       25 

Proteins. — The  proteid  contents  of  the  bacterial  cell  may 
cause  inflammation  and  fever. 

Putrefaction. — When  fermentation  is  accompanied  by 
development  of  offensive  gases,  a  decomposition  occurs,  which 
is  called  putrefaction,  and  this,  in  organic  substances,  is  due 
entirely  to  bacteria. 

Producers  of  Disease. — Various  pathologic  processes  are 
caused  by  bacteria,  the  name  given  to  such  diseases  being 
infectious  diseases,  and  the  germs  themselves  called  disease- 
producing  or  pathogenic  bacteria.  Those  which  do  not  form 
any  pathologic  process  are  called  non-pathogenic  bacteria. 

Ferments  may  be  diastatic,  changing  starch  into  sugar,  or 
proteolytic,  transforming  albumins  into  more  soluble  substances, 
of  which  gelatin  liquefaction  is  an  example.  Inverting,  chang- 
ing a  sugar  from  one  that  does  not  undergo  fermentation  into 
one  that  does. 

Coagulating,  fat-splitting,  hydrolytic  ferments  are  some  of  the 
other  varieties. 

Toxins  and  toxalbumins  are  various  albuminoids  produced 
in  the  animal  organism  and  in  culture-media  which  are  very 
poisonous,  and  are  considered  the  prime  cause  of  disease. 

Pigmentation. — Some  bacteria  are  endowed  with  the 
property  of  forming  pigments  either  in  themselves,  or  pro- 
ducing a  chfomogenic  body  which,  when  set  free,  gives  rise  to 
the  pigment.  In  some  cases  the  pigments  have  been  isolated 
and  many  of  the  properties  of  the  anilin  dyes  discovered  in  them. 

Phosphorescence. — Many  bacteria  have  the  power  to  form 
light,  giving  to  various  objects  which  they  inhabit  a  character- 
istic glow  or  phosphorescence. 

Fluorescence. — An  iridescence,  or  play  of  colors,  develops 
in  some  of  the  bacterial  cultures. 

Gas-formation. — Many  bacteria,  anaerobic  ones  especially, 
produce  gases,  noxious  and  odorless;  in  the  culture-media  the 
bubbles  which  arise  soon  displace  the  media. 

Odors. — Some  germs  form  odors  characteristic  of  them :  some 
are  pleasant  and  even  fragrant;  others,  foul  and  nauseous. 

Effect  of  Age. — With  age,  bacteria  lose  their  strength  and 
die. 


26          ESSENTIALS  OF  BACTERIOLOGY 

CHAPTER  III 
METHODS   OF  EXAMINATION 

WE  divide  the  further  study  of  the  general  characteristics  of 
bacteria  into  two  portions: 

First,  the  examination  of  bacteria  by  aid  of  the  microscope. 

Second,  the  continued  study  through  artificial  cultivation. 

They  both  go  hand  in  hand;  the  one  incomplete  without  the 
other. 

Microscopic. — The  ordinary  microscope  will  not  suffice  for 
bacteriologic  research.  Certain  special  appliances  must  first 
be  added.  It  is  not  so  much  required  to  have  a  picture  very 
large,  as  to  have  it  sharp  and  clear. 

Oil-immersion  Lens. — The  penetration  and  clearness  of  a 
lens  are  very  much  influenced  by  the  absorption  of  the  rays  of 
light  emerging  from  the  picture.  In  the  ordinary  dry  system 
many  of  the  light  rays,  being  bent  outward  by  the  air  which  is 
between  the  object  and  the  lens,  do  not  enter  the  lens,  and  are 
lost.  By  interposing  an  agent  which  has  the  same  refractive 
index  as  glass,  cedar-oil  or  clove-oil,  for  example,  all  the  rays 
of  light  from  the  object  enter  directly  into  the  lens. 

The  "homogeneous  system,"  or  oil-immersion  lens,  consists 
of  a  system  of  lenses  which  can  be  dipped  into  a  drop  of  cedar- 
oil  placed  upon  the  cover-glass,  and  which  is  then  ready  for  use. 

Abbe's  Condenser. — The  second  necessary  adjunct  is  a  com- 
bination of  lenses  placed  under- 
neath the  stage,  for  bringing 
wide  rays  of  light  directly  under 
the  object.  It  serves  to  intensify 
»  the  colored  pictures  by  absorbing 

Fig.  7.— Abbe's  condenser.  or  hiding  the  unstained  structure. 

This  is  very  useful  in  searching 

a  specimen  for  bacteria,  since  it  clears  the  field  of  every- 
thing that  is  not  stained.  It  is  called  Abbe's  condenser 
(Fig.  7).  Together  with  it  is  usually  found  an  instrument 


METHODS   OF   EXAMINATION  27 

for  shutting  off  part  of  the  light — a  blender  or  diaphragm  (Fig. 
8) .  When  the  bacteria  have  been  found,  and  their  relation  to 
the  structure  is  to  be  studied,  the  "Abbe''  is  generally  shut 
out  by  the  iris  blender,  and  the  structure  comes  more  plainly 
into  view.  A  white  light  (daylight  or  a  Welsbach  burner)  is 
best  for  bacterial  study:  use  the  plane  mirror  for  daylight  and 
the  concave  mirror  for  artificial  light. 

For  all  stained  bacteria  the  oil-immersion  lens  and  Abbe  con- 
denser, without  the  use  of  blender.  For  unstained  specimens, 
oil-immersion  and  the  narrowed  blender. 

When  examining  with  low-power  objective,  use  a  strong 
ocular.  When  using  high-power  objective  use  weak  ocular.  A 


Fig.  8. — Iris  blender 

revolving  nose-piece  will  be  found  very  useful,  since  it  is  some- 
times necessary  to  change  the  objective  on  the  same  field,  and 
this  insures  a  great  steadiness  of  the  object. 

Great  cleanliness  is  needed  in  all  bacteriologic  methods,  but 
nowhere  more  so  than  in  the  microscopic  examination. 

The  cover-glass  should  be  very  carefully  washed  in  alcohol, 
and  dried  with  a  soft  linen  rag.  To  remove  the  stains  on  the 
cover-glasses  that  have  been  used,  they  should  be  soaked  in 
hydrochloric  acid  or  placed  in  a  6  per  cent,  aqueous  solution  of 
potassium  dichromate  with  6  per  cent,  of  strong  sulphuric  acid, 
washed  in  water,  and  kept  in  absolute  alcohol. 

Examination  of  Unstained  Bacteria. — As  the  coloring  of 
bacteria  kills  them  and  changes  their  shape  to  some  extent,  it 


28  ESSENTIALS    OF    BACTERIOLOGY 

is  preferable  to  examine  bacteria,  when  possible,  in  their  natu- 
ral state. 

We  obtain  the  bacteria  for  examination  either  from  liquid  or 
solid  media. 

From  Liquids. — With  a  long  platinum  needle  the  end  of 
which  is  bent  into  a  loop  (Fig.  9,  a)  obtain  a  small  drop 
from  the  liquid  containing  the  bacteria,  and  place  it  on  a  cover- 
glass  or  slide,  careful  that  no  bubbles  remain. 

Sterilize  Instruments. — Right  here  we  might  say  that  it  is 
best  to  accustom  one's  self  to  pass  all  instruments,  needles, 
etc.,  through  the  flame  before  and  after  each  procedure;  it 
insures  safety;  and  once  in  the  habit,  it  will  be  done  automati- 
cally. 

From  Solid  Media.— With  a  straight-pointed  platinum 
needle  (Fig.  9,  b)  a  small  speck  of  the  medium  is  taken  and 


Fig.  9. — Platinum  needles  for  transferring  bacteria  made  from  No.  27 
platinum  wire  inserted  in  glass  rods:  a,  Looped  needle;  b,  straight-pointed 
needle  (McFarland) 

rubbed  upon  a  glass  slide  with  a  drop  of  sterilized  water  or 
bouillon,  and  from  this  a  little  is  taken  on  cover-glass,  as  before. 
The  cover-glass  with  its  drop  is  now  placed  on  the  glass  slide, 
carefully  pressing  out  all  bubbles.  Then  a  drop  of  cedar-oil  is 
laid  on  top  of  the  cover-glass,  and  the  oil-immersion  lens  dipped 
gently  down  into  it  as  close  as  possible  to  the  cover-glass,  the 
narrow  blender  shutting  off  the  Abbe  condenser,  for  this  being 
an  unstained  specimen,  we  want  but  little  light.  We  now  apply 
the  eye,  and  if  not  in  focus,  use  the  fine  adjustment  or  the 
coarse,  but  always  away  from  the  object — i.  e.,  toward  us — 
since  the  distance  between  the  specimen  and  the  lens  is  very 
slight,  it  does  not  require  much  turning  to  break  the  cover- 
glass  and  ruin  the  specimen.  Having  found  the  bacterium, 
we  see  whether  it  is  bacillus,  micrococcus,  or  spirillum ;  discover 
if  it  is  motile  or  not.  That  is  about  all  we  can  ascertain  by 
this  method. 


METHODS   OF   EXAMINATION  2 9 

Hanging  Drop  (Fig.  10).— When  the  looped  platinum 
needle  is  dipped  into  a  liquid,  a  very  finely  formed  globule  will 
hang  to  it;  this  can  be  brought  into  a  little  cupped  glass  slide 
(an  ordinary  microscopic  glass  slide  with  a  circular  depression 
in  the  center)  in  the  following  manner:  The  drop  is  first 
brought  upon  a  cover-glass;  the  edges  of  the  concavity  on  the 
glass  slide  are  smeared  with  vaselin,  and  the  slide  inverted 
over  the  drop;  the  cover-glass  sticks  to  the  smeared  slide, 
which,  when  turned  over,  holds  the  drop  in  the  depression 
covered  by  the  cover-glass,  thus  forming  an  air-tight  cell;  here 
the  drop  cannot  evaporate.  Both  slide  and  cover-glass  should 
first  be  sterilized  by  heat. 


Fig.  10. — A  "concave  slide"  with  "hanging  drop"  (McFarland). 

Search  for  the  bacteria  with  a  weak  lens;  having  found  them, 
place  a  drop  of  cedar-oil  upon  the  cover-glass,  and  bring  the  oil 
immersion  into  place  (here  is  where  a  nose-piece  comes  in  very 
useful) ,  careful  not  to  press  against  the  cell,  for  the  cover-glasses 
are  very  fragile  in  this  position. 

Search  the  edges  of  the  drop  rather  than  the  middle;  the  bac- 
teria will  usually  be  very  thick  in  the  center  and  not  so  easily 
distinguished. 

Spores,  automatic  movements,  fission,  and  cultivation  in 
general  can  be  studied  for  several  days.  This  moist  chamber 
can  be  placed  in  a  brood-oven  or  on  the  ordinary  warming 
stages  of  the  microscope. 

Agglutination  as  observed  in  WidaPs  test  is  best  seen  in  the 
hanging  drop. 


30  ESSENTIALS    OF    BACTERIOLOGY 

CHAPTER  IV 
STAINING   OF  BACTERIA 

STAINING  or  coloring  bacteria  is  done  in  order  to  make  them 
prominent  and  to  obtain  permanent  specimens.  It  is  also 
necessary  to  bring  out  the  structure  of  the  bacteria,  and 
serves  in  many  instances  as  a  means  of  diagnosis;  it  would  be 
well-nigh  impossible  to  discover  them  in  the  tissues  without 
staining. 

Anilin  Colors. — Of  the  numerous  dyes  in  the  market,  nearly 
all  have,  at  one  time  or  other,  been  used  in  staining  bacteria. 
But  now  only  a  very  few  find  general  use,  and  with  methylene- 
blue  and  fuchsin  nearly  every  object  can  be  accomplished. 

Basic  and  Acid  Dyes. — Ehrlich  was  the  first  to  divide  the 
anilin  dyes  into  two  groups,  the  basic  colors  to  which  belong — 
Gentian-violet,  or  pyoktanin.  Basic  fuchsin. 

Methyl-violet,  or  dahlia.  Bismarck-brown. 

Methylene-blue  (not  methyl  blue).    Thionin. 

Safranin. 
And  the  acid  colors  to  which  eosin  and  acid  fuchsin  belong. 

The  basic  dyes  stain  the  bacteria  and  the  nuclei  of  cells;  the 
acid  dyes  stain  chiefly  the  tissue,  leaving  the  bacteria  almost 
untouched.  Carmin  and  hematoxylin  are  also  useful  as  con- 
trast stains,  affecting  bacteria  very  slightly.  The  anilin  dyes 
are  soluble  in  alcohol  or  water  or  a  mixture  of  the  two. 

Staining  Solutions.— A  saturated  solution  of  the  dye  is 
made  with  alcohol.  This  is  called  the  stock  or  concentrated 
solution;  i  part  of  this  solution  to  about  100  parts  of  distilled 
water  constitutes  the  ordinary  aqueous  solution  in  use  or  weak 
solution. 

It  is  readily  made  by  adding  to  an  ounce  bottle  of  distilled 
water  enough  of  the  strong  solution  until  the  fluid  is  still  opaque 
in  the  body  of  the  bottle,  but  clear  in  the  neck  of  the  same. 

These  weak  solutions  should  be  renewed  every  three  or  four 
weeks,  otherwise  the  precipitates  formed  will  interfere  with  the 
staining. 


STAINING    OF   BACTERIA  31 

Compound  Solutions. — By  means  of  certain  chemical  agents 
the  intensity  of  the  anilin  dyes  can  be  greatly  increased. 

Mordants. — Agents  that  "bite"  into  the  specimen,  carrying 
the  stain  with  them,  depositing  it  in  the  deeper  layers,  are 
called  mordants  or  etchers. 

Various  metallic  salts  and  vegetable  acids  are  used  for  such 
purpose. 

The  mother  liquid  of  the  anilin  dyes,  anilin-oil^  a  member  of 
the  aromatic  benzol  group,  has  also  this  property. 

Anilin-oil  Water. — Anilin-oil  is  shaken  up  with  water  and 
then  filtered;  the  anilin  water  so  obtained  is  mixed  with  the 
dyes,  forming  the  ''anilin-water  gentian-violet"  or  anilin-water 
fuchsin,  etc. 

Carbol-fuchsin. — Carbolic  acid  or  phenol  can  be  used 
instead  of  anilin-oil,  and  forms  one  of  the  main  ingredients  of 
Ziehl's  or  Neelsen's  solution,  used  principally  in  staining  Bacil- 
lus tuberculosis.  Kiihne  has  a  carbol-methylene-blue  made 
similar  to  the  carbol-fuchsin. 

Alkaline  Stains. — Alkalis  have  the  same  object  as  the  above 
agents,  namely,  to  intensify  the  picture.  Potassium  hydroxid, 
ammonium  carbonate,  and  sodium  hydroxid  are  used. 

Lofrler's  alkaline  blue  and  Koch's  weak  alkaline  blue  have 
in  them  potassium. 

Heat. — Warming  or  boiling  the  stains  during  the  process  of 
staining  increases  their  intensity. 

Decolorizing  Agents. — The  object  after  staining  is  usually 
overcolored  in  some  part,  and  then  decolorizing  agents  are  em- 
ployed. Water  is  sufficient  in  many  cases;  alcohol  and  strong 
mineral  acids  combined  are  necessary  in  some. 

lodin  as  Used  in  Gram's  Method. — Belonging  to  this  group, 
but  used  more  in  the  sense  of  a  protective,  is  tincture  of  iodin. 
It  picks  out  certain  bacteria,  which  it  coats;  prevents  them  from 
being  decolorized,  but  allows  all  else  to  be  faded.  Then,  by 
using  one  of  the  acid  or  tissue  dyes,  a  contrast  color  or  double 
staining  is  obtained.  Many  of  the  more  important  bacteria  are 
not  acted  upon  by  the  iodin,  and  it  thus  becomes  a  very  useful 
means  of  diagnosis. 


32  ESSENTIALS    OF    BACTERIOLOGY 

FORMULAS    OF    DIFFERENT    STAINING    SOLUTIONS 
I.     Saturated  Alcoholic  Solution. 

Place  about  10  grams  of  the  powdered  dye  in  a  bottle  and 
add  40  grams  of  alcohol.  Shake  well  and  allow  to  settle. 
This  can  be  used  as  the  stock  bottle. 

II.     Weak  Solutions. 

Made  by  adding  about  i  part  of  stock  solution  (I)  to  10 
parts  of  distilled  water.  This  is  the  ordinary  solution  in  use. 

III.    Anilin-oil  Water. 

Anilin-oil 5  parts. 

Distilled  water 100      "    — M. 

Shake  well  and  filter.     To  be  made  fresh  each  time. 

IV.    Anilin-water  Dyes. 

Saturated  alcoholic  solution  of  the  dye  n  parts. 

Anilin-oil  water 100 

Absolute  alcohol 10      "     — M. 

Can  be  kept  ten  days. 

V.    Alkaline  Methylene-blue. 

A.  Loffler's: 

Saturated  alcoholic  solution  methylene- 

blue 3°-° 

Solution  potassium  hydroxid  (i :  10,000)   100.0. — M. 

B.  Koch's: 

Solution  potassium  hydroxid  (10  per 
cent.) 0.2 

Saturated  alcoholic  solution  methylene- 
blue i.o 

Distilled  water  .  .  200.0.— M. 


STAINING   OF   BACTERIA  33 

VI.     Phenol  Solutions. 

A.  Ziehl-Neelsen: 

Fuchsin  (powdered)  i  part. 

Alcohol 10  parts. 

5  per  cent,  solution  phenol 100      "     — M. 

Filter.     The  older  the  solution,  the  better. 

B.  Kiihne: 

Methylene-blue 1.5 

Alcohol 10.0 

5  per  cent,  solution  phenol 100.0 

Add  the  acid  gradually.     This  solution  loses  strength  with 
age. 

VII.    Gram's  lodin  Solution. 

lodin i.o 

Potassium  iodid ! 2.0 

Distilled  water 300.0 — M. 

VIII.    Loffler's  Mordant  (for  Flagella). 

Aqueous    solution  of    tannin    (20    per 

cent.)  ....*. 10  parts. 

Aqueous  solution  ferric  sulphate  (5  per 

cent.) - i  part. 

Aqueous  decoction  of  logwood  (i  :  8) .  .       4  parts. — M. 
Keep  in  well-corked  bottle. 

IX.     llnna's  Borax  Methyl-blue. 

Borax i  part. 

Methyl  blue i  part. 

Water 100  parts.— M. 

X.    Gobbet's  Acid  Blue  (Rapid  Stain). 

Methylene-blue 2.0 

25  per  cent,  sulphuric  acid 100.0 — M. 

3 


34  ESSENTIALS    OF    BACTERIOLOGY 

XI.    Alkaline  Anilin-water  Solutions. 

Sodium  hydroxid  (i  per  cent.) i.o 

Anilin-oil  water 100.0 — M. 

And  add — 

Fuchsin,  or  methyl- violet  powdered  .  .  .       4.0 

Cork  well.     Filter  before  using. 

XII.     Roux's  Double  Stain. 

Dahlia  or  gentian-violet 0.5  gm. 

Methyl-green 1.5     " 

Distilled  water 200.0    "  — M. 

Use  as  other  stains,  without  acid. 

XIII.     Neisser's  Stain  (jor  Diphtheria). 

Solution  I. 

Methylene-blue i  gm. 

Alcohol  (96  per  cent.)   20  c.c. 

Dissolve  and  add — 

Water 950  c.c. 

Glacial  acetic  acid 50  c.c. — M. 

Solution  II. 

Vesuvin 2  gm. 

Water 1000  c.c. — M. 

Stain  cover-glasses — (i)  Three  seconds  in  solution  I;  (2) 
wash  in  water;  (3)  three  seconds  in  No.  2;  (4)  wash  in  water. 
Body  of  bacillus,  brown;  oval  granules  at  each  end,  blue. 

XIV.     Carbol-thionin  (Nicolle). 

Saturated  solution  thionin  in  alcohol  (90 

per  cent.) 10  c.c. 

Aqueous  solution  phenol  (i  per  cent.) .  .    100  c.c. — M. 
Stain  sections  one-half  to  one  minute. 


STAINING   OF   BACTERIA  35 

XV.    Capsule  Stain  of  Hiss. 

Use  the  following,  heated  until  it  steams: 

Saturated  alcoholic  solution  of  gentian- 
violet  or  fuchsin 5  c.c. 

Distilled  water 95  c.c. 

Wash  in  20  per  cent,  solution  of  cupric  sulphate  crystals. 

XVI.    Capsule  Stain  of  Welch. 

(i)  Pour  glacial  acetic  acid  on  film.  After  a  few  seconds 
replace  with  anilin-water  gentian-violet  without  washing  in 
water.  (2)  Remove  all  acid  by  several  additions  of  stain,  and 
allow  it  to  act  for  three  to  four  minutes.  (3)  Wash  and  examine 
in  salt  solution  0.8-2.0  per  cent. 

XVII.     Romanowsky  Stains. 

A  compound  dye  originally  used  for  malarial  parasites,  but 
now  employed  in  some  of  its  modifications  in  staining  blood- 
films,  bacteria  in  tissues,  and  protozoa  generally. 

The  stain  is  difficult  to  prepare,  and  can  be  purchased  of 
supply  houses  to  better  advantage. 

The   chief   modifications  are: 

Leishman's  stain,  consisting  of  a  i  per  cent,  solution  methyl- 
ene-blue,  to  which  0.5  per  cent,  sodium  carbonate  has  been 
added  and  allowed  to  stand  for  twelve  hours  in  incubator  at 
65°  C.,  and  then  ten  days  at  room  temperature,  and  a  solution 
of  eosin  (i  :  1000)  in  water.  Equal  parts  of  these  solutions 
are  mixed  and  allowed  to  stand  for  six  hours.  After  it  has 
been  washed  and  dried,  the  precipitate  is  dissolved  in  methyl- 
alcohol. 

Giemsa  stain: 

Azur  II. — eosin 3  parts. 

Azur  II 8 

Glycerin  (pure)  250       " 

Methyl-alcohol '. 250       "      — M. 

Azur  is  a  mixture  of  methylene-blue  and  eosin  prepared  in  a 
special  way. 


36  ESSENTIALS    OF    BACTERIOLOGY 

Jenner's  Stain. — See  page  189. 

/.  H.  Wright's  stain:  made  in  much  the  same  way  as 
Irishman's.  The  precipitate  is  not  washed,  but  the  saturated 
methyl-alcohol  solution  is  filtered  and  further  diluted  with 
methyl-alcohol.  The  stains  are  used  in  very  dilute  form. 
Where  the  blood-films  or  exudates  are  not  first  fixed  in  alcohol, 
the  concentrated  stain  is  allowed  to  cover  the  preparation  for 
five  to  twenty  seconds  to  fix;  then  water  is  poured  on  to  dilute 
and  from  five  to  fifteen  minutes  allowed  for  staining,  the  excess 
removed  with  water.  The  stains  can  be  purchased  in  powder 
form,  and  need  only  be  mixed  with  water  to  be  ready  for  use. 


CHAPTER  V 
GENERAL  METHOD  OF  STAINING  SPECIMENS 

Cover-glass  Preparations.— The  material  is  evenly  spread 
in  as  thin  a  layer  as  possible  upon  a  cover-glass;  then,  to  spread 
it  still  more  finely,  a  second  cover-glass  is  pressed  down  upon 
the  first  and  the  two  slid  apart.  This  also  secures  two  speci- 
mens. Before  they  can  be  stained,  they  must  be  perfectly  dry, 
otherwise  deformities  will  arise  in  the  structure. 

Drying  the  Specimen.— The  cover-glass  can  be  set  aside  to 
dry,  or  held  in  the  fingers  over  the  Bunsen  burner  (the  fingers 
preventing  too  great  a  degree  of  heat).  Since  most  of  the 
specimens  contain  a  certain  amount  of  albuminoid  material, 
it  is  best  in  all  cases  to  "fix" — i.  e.,  to  coagulate  the  albumin. 
This  is  accomplished  by  passing  the  cover-glass  (after  the  speci- 
men is  dry)  three  times  through  the  flame  of  the  burner,  about 
three  seconds  being  consumed  in  doing  so,  the  glass  being  held 
in  a  small  forceps,  smeared  side  up. 

The  best  forceps  for  grasping  cover-glasses  is  a  bent  one,  bent 
again  upward,  near  the  ends  (Fig.  1 1) .  It  prevents  the  flame 
or  staining  fluid  from  reaching  the  fingers. 


GENERAL  METHOD  OF  STAINING   SPECIMENS  37 

The  object  is  now  ready  for  staining. 

Staining. — A  few  drops  of  the  staining  solution  are  placed 
upon  the  cover-glass  so  that  the  whole  specimen  is  covered, 
and  is  left  on  a  few  minutes,  the  time  depending  upon  the 
variety,  the  strength  of  stain,  and  the  object  desired.  Instead 
of  placing  the  dye  upon  the  object,  the  cover-glass  can  be 
immersed  in  a  small  glass  dish  containing  the  solution;  or,  if 
heat  is  desired  to  intensify  or  hasten  the  process,  a  watch- 
crystal  holding  the  stain  is  placed  over  a  Bunsen  burner  and 
in  it  the  cover-glass;  the  cover-glass  may  be  held  directly  in 
the  flame  with  the  staining  fluid  upon  it,  which  must  be 
constantly  renewed  until  the  process  is  completed,  or  the 
cover-glass  can  be  heated  in  a  test-tube,  containing  stain 
solution. 


Fig.  n. — Author's  bent  forceps  for  holding  cover-glass  over  flame. 

Removing  Excess  of  Stain. — The  surplus  stain  is  washed 
off  by  dipping  the  glass  in  distilled  water. 

The  water  is  removed  by  drying  between  filter-paper  or 
simply  allowed  to  run  off  by  standing  the  cover-glass  slant- 
wise against  an  object.  When  the  specimen  is  to  be  examined 
in  water  (which  is  always  best  with  the  first  preparation  of 
the  specimen,  as  the  Canada  balsam  destroys  to  some  extent 
the  natural  appearance  of  the  bacteria),  a  small  drop  of  ster- 
ilized water  is  placed  upon  the  glass  slide,  and  the  cover-glass 
dropped  gently  down  upon  it,  so  that  the  cover-glass  remains 
adherent  to  the  slide. 

The  dry  system  or  the  oil  immersion  can  now  be  used. 

When  the  object  has  been  sufficiently  examined,  it  can  be 
permanently  mounted  by  lifting  the  cover-glass  off  the  slide 
(this  is  facilitated  by  letting  a  little  water  flow  under  it,  one  end 


38  ESSENTIALS    OF    BACTERIOLOGY 

being  slightly  elevated) .  The  water  that  still  adheres  is  dried 
off  in  the  air  or  gently  over  the  flame,  and  when  perfectly  dry, 
it  is  placed  upon  the  drop  of  Canada  balsam  which  has  been  put 
upon  the  glass  slide. 

In  placing  the  cover-glass  in  the  staining  solutions  one  must 
be  careful  to  remember  which  is  the  spread  side,  by  holding  it 
between  oneself  and  the  window  and  scraping  the  sides  care- 
fully with  the  sharp  point  of  the  forceps,  the  side  having  the 
specimen  on  it  will,  show  the  marks  of  the  instrument. 

Little  glass  dishes,  about  one-half  dozen,  should  be  at  hand 
for  containing  the  various  stains  and  decolorants. 

Tissue  Preparations.— In  order  to  obtain  suitable  specimens 
for  staining,  very  thin  sections  of  the  tissue  must  be  made. 

As  with  histologic  preparations,  the  tissue  must  be  hardened 
before  it  can  be  cut  thin  enough.  Alcohol  is  the  best  agent  for 
this  purpose. 

Pieces  of  the  tissue  one-quarter  inch  in  size  are  covered  with 
alcohol  for  twenty-four  to  forty-eight  hours. 

When  hardened,  it  must  be  fixed  upon  or  in  some  firm  object. 
A  paste  composed  of — 

Gelatin i  part. 

Glycerin 4  parts. 

Water 2      " , 

will  make  it  adhere  firmly  to  a  cork  in  about  two  hours,  or  it  can 
be  embedded  in  a  small  block  of  paraffin,  and  covered  over  with 
melted  paraffin.  Celloidin  may  be  used  as  an  embedding  agent, 
and  formalin  is  useful  to  harden  tissue  quickly. 

Cutting. — The  microtome  (Fig.  12)  should  be  able  to  cut 
sections  5-^  inch  in  thickness;  this  is  the  fineness  usually 
required. 

The  sections  are  brought  into  alcohol  as  soon  as  cut,  unless 
they  have  been  embedded  in  paraffin,  when  they  are  first  washed 
in  chloroform  to  dissolve  out  the  paraffin. 

Staining. — All  the  various  solutions  should  be  in  readiness, 
best  placed  in  the  little  dishes  in  the  order  in  which  they  are  to 


GENERAL   METHOD   OF    STAINING   SPECIMENS 


39 


be  used,  as  a  short  delay  in  one  of  the  steps  may  spoil  the  speci- 
men. 

A  very  useful  instrument  for  transferring  the  delicate  sections 
from  one  solution  to  another  is  a  little  metal  spatula,  the  blade 
being  flexible  (Fig.  13). 


Fig.   12. — Large  laboratory  microtome  (Mallory  and  Wright). 

A  still  better  plan,  especially  when  the  tissue  is  " crumbling," 
is  to  carry  out  the  whole  procedure  on  the  glass  slide. 


Fig.   13.— Spatula  for  lifting  sections. 

General  Principles.— The  section  is  transferred  from  the 
alcohol  in  which  it  has  been  kept  into  water,  which  removes 
the  excess  of  alcohol,  from  here  into — 

Dish  /,  containing  the  stain,  where  it  remains  five  to  fifteen 
minutes.  Then — 


40  ESSENTIALS    OF    BACTERIOLOGY 

Dish  II,  containing  5  per  cent,  acetic  acid  (i  :  20),  where  it 
remains  one-half  to  one  minute.  The  acid  removes  the  excess 
of  stain. 

Dish  III,  water,  to  rinse  off  the  acid.  The  section  can  now 
be  placed  under  the  microscope,  covered  with  cover-glass  to  see 
if  the  intensity  of  the  stain  is  sufficient  or  too  great.  A  second 
section  is  then  taken,  avoiding  the  errors,  if  any;  and  having 
reached  this  stage,  proceeded  with  as  follows: 

Dish  IV,  alcohol,  two  to  three  seconds,  to  remove  the  water  in 
the  tissue. 

V.  A  few  drops  of  oil  of  cloves,  just  long  enough  to  clear  the 
specimen  to  make  it  transparent  (so  that  an  object  placed 
underneath  will  shine  through). 

VI.  Remove  excess  with  filter-paper. 

VII.  Mount  in  Canada  balsam  (xylol  balsam). 


CHAPTER  VI 
SPECIAL  METHODS  OF  STAINING  AND  MODIFICATIONS 

Gram's  Method  of  Double  Staining  (For  Cover-glass 
Specimens). — I.  A  hot  solution  of  anilin- water  gentian- violet 
two  to  ten  minutes. 

II.  Directly,  without  washing,  into  Gram's  solution  of  iodin 
potassium-iodid  one  to  three  minutes  (the  cover-glass  looks 
black). 

III.  Wash  in  alcohol  60  per  cent,  until  only  a  light  brow 
shade  remains  (as  if  the  glass  were  smeared  with  dried  blood) . 

IV.  Rinse  off  alcohol  with  water. 

V.  Contrast  color  with  either  eosin,  picrocarmin,  or  Bis- 
marck-brown.    The  bacteria  will  appear  deep  blue,  all  else 
red  or  brown  on  a  very  faint  brown  background. 


SPECIAL   METHODS   OF   STAINING   AND   MODIFICATIONS      41 

Gram's  Method  for  Tissues  (Modified  byGunther}: 

I.  Stain  in  anilin-water  gentian- violet .  .  .      i  minute. 
II.  Dry  between  filter-paper. 

III.  lodin  potassium  iodid  solution 2  minutes. 

IV.  Alcohol \  minute. 

V.  3  per  cent,  solution  hydrochloric  acid 

in  alcohol 10  seconds. 

VI.  Alcohol,  oil  of  cloves,  and  Canada  balsam. 

Behavior  of  the  More  Important  Bacteria  to  Gram's  Stain. — 
Positive  means  that  the  bacteria  retain  the  primary  color,  or 
gentian-violet,  negative  that  they  do  not. 

Positive.  Negative. 

Tubercle  bacillus.  Colon  bacillus. 

Smegma  bacillus.  Typhoid  bacillus. 

Lepra  bacillus.  Cholera  bacillus. 

Anthrax  bacillus.  Influenza  bacillus. 

Tetanus  bacillus.  Friedlander's  bacillus. 

Diphtheria  bacillus.  Plague  bacillus. 

Pneumococcus.  Diplococcus  intracellularis. 

Streptococcus.  Gonococcus. 

Staphylococcus.  Koch-Weeks'  bacillus. 

Cocci  of  the  urethra.  Conjunctivitis  bacillus  of  Morax. 

To  Stain  Spores. — Since  spores  have  a  very  firm  capsule, 
which  tends  to  keep  out  all  external  agents,  a  very  intensive 
stain  is  required  to  penetrate  them,  but  once  this  object  is 
attained,  it  is  equally  as  difficult  to  decolorize  them. 

A  cover-glass  prepared  in  the  usual  way,  i.  e.,  drying  and 
passing  the  specimen  through  the  flame  three  times,  is  placed  in 
a  watch-crystal  containing  ZiehPs  carbol-fuchsin  solution,  and 
the  same  placed  upon  a  rack  over  a  Bunsen  burner,  where  it  is 
kept  at  boiling-point  for  one  hour,  careful  to  supply  fresh  solution 
at  short  intervals  lest  it  dry  up. 

The  bacilli  are  now  decolorized  in  alcohol  containing  J  per 
cent,  hydrochloric  acid.  A  contrast  color,  preferably  methyl- 
ene-blue,  is  added  for  a  few  minutes. 


42          ESSENTIALS  OF  BACTERIOLOGY 

The  spores  will  appear  as  little  red  beads  in  the  blue-stained 
bacteria,  and  loose  spores  lying  about  outside  the  cell-wall. 

Spore  Stain  (Modified) . — I.  Carbol-fuchsin  on  cover-glass  and 
heated  in  the  flame  to  boiling-point  20  to  30  times. 

II.  25    per  cent,    sulphuric   acid,   two   seconds;   rinsed  in 
water. 

III.  Methylene-blue  contrast. 

Alex.  Klein  recommends  the  following  spore  method:  mix  a 
little  of  the  culture  (potato)  with  three  drops  of  physiologic  salt 
solution,  and  heat  gently  with  an  equal  quantity  of  carbol- 
fuchsin  for  a  period  of  six  minutes.  Spread  then  on  cover- 
glasses,  dry  in  the  air,  and  fix  by  passing  three  times  through 
Bunsen-burner  flame.  Decolorize  in  i  per  cent,  sulphuric  acid 
for  one  to  two  seconds;  contrast  in  weak  methylene-blue. 

Bowhill's  Orcein  Stain. 

Saturated  alcoholic  solution  of  orcein  .  .  15  c.c. 

20  per  cent,  aqueous  solution  tannin ...  TO  c.c. 

Distilled  water 30  c.c. — M. 

Filter. 

Use  orcein  solution  in  watch-glass,  float  cover-glass  in  it,  and 
heat  gently,  not  boil,  for  ten  minutes.  Wash  in  water.  Dry 
and  mount  in  balsam. 

Five  per  cent,  chromium  trioxid  applied  for  fifteen  minutes 
has  been  recommended  in  staining  spores.  This  is  followed 
by  the  carbol-fuchsin  stain  as  above. 

Sporogenic  bodies  stain  quite  readily,  and  in  order  to  distin- 
guish them  from  spores  Ernst  uses  alkaline  methylene-blue, 
slightly  warmed.  Then  rinse  in  water.  Contrast  with  cold 
Bismarck-brown. 

The  spores  are  colored  bright  blue,  the  spore  granules  a  dirty 
blue,  being  mixed  with  the  brown,  which  colors  also  the  bacteria. 

Kiihne's  Method. — In  sections  the  alcohol  used  sometimes 
decolorizes  too  much.  To  obviate  this Kuhne  mixes  the  alcohol 
with  the  stain,  so  that  while  the  section  is  being  anhydrated,  it 
is  constantly  supplied  with  fresh  dye. 


SPECIAL   METHODS   OF   STAINING   AND   MODIFICATIONS      43 

Weigert  uses  anilin-oil  to  dehydrate  instead  of  alcohol,  and 
here,  too,  it  can  be  used  mixed  with  the  dye. 

Capsule  Stain  (Buerger). — I.  Spread  culture  by  means  of  a 
drop  of  ascitic  fluid  on  cover-glass. 

II.  Fix  in  Miiller's  fluid,  which  has  been  saturated  with  5 
per  cent,  bichlorid  of  mercury,  and  warm  for  three  seconds. 

III.  Wash  quickly  in  water;  rinse  in  alcohol. 

IV.  Cover  with  tincture  of  iodin  for  one  minute. 

V.  Wash  in  alcohol  and  dry  in  air. 

VI.  Stain  in  anilin-water  gentian-violet  for  two  seconds. 

VII.  Wash  in  2  per  cent,  salt  solution. 

VIII.  Mount  in  salt  solution  ringed  with  vaselin. 

Flagella  Stain,  with  Lo frier's  Mordant.— I.  A  few  drops 
of  the  mordant  (No.  VIII,  p.  33)  are  placed  upon  the  spread 
cover-glass  and  heated  until  it  steams. 

II.  Wash  with  water  until  the  cover-glass  looks  almost  clean, 
using  a  small  piece  of  filter-paper  to  rub  off  the  crusts  which 
have  gathered  around  the  edges. 

III.  Anilin-water  fuchsin  (neutral)  held  in  flame  about  one 
and  one-half  minutes. 

IV.  Wash  in  water. 

If  the  stain  is  properly  made,  the  bacteria  are  deeply  colored 
and  the  flagella  seen  as  little  dark  lines  attached  to  them. 

Unna's  Method  for  Fungi. — Especially  useful  for  epidermic 
scales.  Moisten  horny  scale  or  crust  with  acetic  acid;  mace- 
rate between  two  glass  slides;  dry  in  flame;  wash  out  fat  with 
ether  and  alcohol  (equal  parts) ;  stain  in  borax  methyl  blue  for 
ten  seconds  (over  flame) ;  bleach  with  glycerin  and  ether  (equal 
parts) ;  rinse  in  water,  alcohol,  dry  and  mount. 


44  ESSENTIALS    OF    BACTERIOLOGY 

CHAPTER  VII 
METHODS  OF  CULTURE 

Artificial  Cultivation. — The  objects  of  cultivation  are  to 
obtain  germs  in  pure  culture,  free  from  all  foreign  matter, 
isolated,  and  so  developed  as  to  be  readily  used  either  for 
microscopic  examination  or  animal  experimentation. 

To  develop  bacteria  properly  we  supply,  as  nearly  as  possi- 
ble, the  conditions  which  hold  for  the  especial  germ  in  nature. 
With  the  aid  of  solid  nutrient  media  the  bacteria  can  be  easily 
separated,  and  the  methods  are  nearly  perfect. 

Sterilization  of  Cultures. — If  we  place  our  nutrient  mate- 
rial in  vessels  that  have  not  been  properly  disinfected,  we  will 
obtain  growths  of  bacteria  without  having  sown  any. 

If  we  have  thoroughly  cleaned  our  utensils  and  then  not  taken 
care  to  protect  them  from  further  exposure,  the  germs  we  have 
sown  will  be  effaced  or  contaminated  by  multitudes  of  others 
that  are  constantly  about  us.  We,  therefore,  have  two  neces- 
sary precautions  to  take: 

First,  thoroughly  to  clean  and  sterilize  every  object  that 
enters  into,  or  in  any  way  comes  in  contact  with,  the  culture. 

Second,  to  maintain  this  degree  of  sterility  throughout  the 
whole  course  of  the  growth,  and  prevent,  by  proper  containers, 
the  entrance  of  foreign  germs. 

Disinfectants. — Corrosive  sublimate  (bichlorid  of  mercury), 
which  is  the  most  effective  agent  we  possess,  cannot  be  generally 
used  because  it  renders  the  soil  unproductive,  and,  therefore, 
must  be  employed  only  in  washing  dishes,  to  destroy  the  old 
cultures.  Even  after  washing  a  few  drops  of  the  solution  may 
remain  and  prevent  growth,  so  that  one  must  be  careful  to  have 
the  glassware  that  comes  in  contact  with  the  nutrient  media 
free  from  the  sublimate. 

Heat. — Heat  is  the  best  agent  we  possess  for  general  use. 
Dry  heat  and  moist  heat  are  the  two  forms  employed,  but  these 
differ  greatly  in  effectiveness.  Thus  Koch  found  that  while 


METHODS   OF   CULTURE 


45 


moist  heat  at  100°  C.  killed  the  spores  of  the  anthrax  bacillus 
in  one  hour,  it  required  three  hours  of  dry  heat  at  140°  C.  to 
produce  death. 

For  obtaining  dry  heat — that  is,  a  temperature  of  150°  C. 
(about  300°  F.) — a  sheet-iron  oven  (Fig.  14)  is  used,  which  can 
be  heated  by  a  gas-burner.  If  it  have  double  walls  (air  circulat- 
ing between),  the  desired  temperature  is  much  more  quickly 
obtained.  A  small  opening  in  the  top  to  admit  a  thermometer 
is  necessary.  These  chests  are  usually  about  i  foot  high, 
i  \  feet  wide,  and  f  foot  deep.  In  them  glassware,  cotton,  and 


Fig.  14. — Hot  air  oven. 

paper  can  be  sterilized.  When  the  cotton  is  turned  slightly 
brown,  it  usually  denotes  sufficient  sterilization.  All  instru- 
ments, where  practicable,  should  be  drawn  through  the  flame 
of  an  alcohol  lamp  or  Bunsen  burner.  One  hour  in  the  oven 
at  170°  C.  usually  sterilizes  glassware,  while  the  ordinary 
germs  in  liquids  may  be  killed  by  boiling  for  five  minutes  if 
no  spores  are  present.  The  boiling  of  any  fluid  at  100°  C. 
for  one  and  one-half  hours  nearly  always  insures  sterilization. 

Moist  Heat.— Steam   at    100°  C.  in   circulation   has   been 
shown  to  be  a  very  effective  application  of  heat. 


46 


ESSENTIALS    OF    BACTERIOLOGY 


Koch's  Steam-chest  (Fig.  15). — Circulating  steam  is  obtained 
by  aid  of  Koch's  apparatus.  This  consists  of  a  cylindric  tin 
chest  about  2j  feet  high  and  about  J  foot  in  diameter;  divided 
in  its  interior  by  a  perforated  diaphragm,  a,  an  upper  cham- 


Fig.  15. — Koch's  steam-chest. 


Fig.   16. — Arnold's  steam  sterilizer. 


ber  for  the  steam,  c,  and  a  lower  one  for  water,  b.  Two  or 
more  gas-burners  placed  underneath  the  chest,  which  stands  on 
a  tripod,  supply  the  heat.  In  the  cover  is  an  opening  for  a  ther- 
mometer. The  chest  is  usually  covered  with  felt.  When  the 
thermometer  registers  100°  C.,  the  culture-medium  or  other  sub- 


METHODS   OF   CULTURE 


47 


stance  to  be  sterilized  is  placed  in  the  steam  and  kept  there 
from  ten  to  fifteen  minutes,  or  longer,  as  required. 

Arnold's  steam  sterilizer  (Fig.  16)  will  answer  every  purpose 
of  the  Koch  steam-chest.  It  is  cheaper,  also  requiring  less 
fuel  to  keep  it  going.  The  steam  does  not  escape,  but  is  con- 
densed in  the  outer  chamber. 

The  autoclave  (Fig.  17),  which  produces  steam  under  pres- 
sure and  allows  a  temperature  of  120°  C.  to  be  obtained,  is  a 
most  effective  method  of  steriliza- 
tion, but  the  higher  temperatures 
are  not  suitable  for  gelatin  or  sugar 
solution.  Gelatin  loses  its  power 
of  solidifying  if  the  boiling  is  pro- 
longed. 

Instead  of  sterilizing  for  a  long 
time  at  once,  successive  sterilization 
is  practised  with  nutrient  media, 
so  that  the  albumin  will  not  be 
too  strongly  coagulated.  Fifteen 
minutes  each  day  for  three  days 
in  succession  in  the  steam-chest  or 
autoclave  is  sufficient. 

Fractional  Sterilization  of 
Tyndall. — Granted  that  so  many 
spores  originally  exist  in  the  object 
to  be  sterilized,  it  is  subjected  to 
60°  C.  for  four  hours,  in  which  time 
a  part  at  least  of  those  spores  have 
developed  into  bacteria,  and  the 
bacteria  destroyed  by  the  further 
application  of  the  heat.  The  next 
day  more  bacteria  will  have  formed, 
and  four  hours'  subjection  to  60°  C. 
heat  will  destroy  them,  and  so,  at  the  end  of  a  week,  using 
four  hours'  application  each  day,  all  the  spores  originally 
present  will  have  germinated  and  the  bacteria  be  destroyed. 

As  modified,  and  in  use  in  most  laboratories,  fifteen  minutes' 


Fig.  17. — Modern  autoclave. 


48  ESSENTIALS    OF    BACTERIOLOGY 

sterilization  in  steam,  at  100°  C.,  on  three  successive  days,  has 
been  found  sufficient  for  nearly  all  purposes,  while  one  sterili- 
zation in  the  autoclave  at  110°  C.  for  fifteen  minutes  will 
serve  in  some  cases,  especially  if  the  media  is  for  immediate 
use. 

Cotton  Plugs  or  Corks. — All  the  glass  vessels  (test-tubes, 
flasks,  etc.)  must  be  closed  with  cotton  plugs,  the  cotton  being 
easily  sterilized  and  preventing  the  entrance  of  germs. 


Fig.  1 8.— Wire  cage. 


Fig.  19. — Cotton-plugged  test-tubes. 


Tin-foil  may  be  used  to  cover  the  cotton,  or  caps  made  of 
india-rubber. 

Test-tubes. — New  test-tubes  are  washed  with  hydrochloric 
acid  and  water  to  neutralize  the  alkalinity  often  present  in 
fresh  glass.  They  are  then  well  washed  and  rubbed  with  a 
brush,  placed  obliquely  to  drain,  and  when  dry,  corked  with 
cotton  plugs.  Then  put  in  the  hot-air  oven  (little  wire  cages 
being  used  to  contain  them)  for  fifteen  minutes,  after  which  they 
are  ready  to  be  filled  with  the  nutrient  media.  (The  cotton 
should  fit  firmly  in  the  tube  and  extend  a  short  space  beyond  it.) 


NUTRIENT   MEDIA  49 

Test-tubes  without  flaring  edges  are  more  desirable,  since  the 
edges  can  easily  be  drawn  out  so  as  to  seal  the  tube. 

Instead  of  test-tubes,  ordinary  3-ounce  panel  medicine  bottles 
can  be  used  for  retaining  the  nutrient  media  and  cultures. 

According  to  late  investigations,  the  glass  tubes  become  suffi- 
ciently sterile  in  the  steam-chest  without  the  preliminary  sterili- 
zation in  the  dry  oven. 


CHAPTER  VIII 
NUTRIENT  MEDIA 

OF  the  many  different  media  recommended  and  used  since 
bacteriology  became  a  science,  we  can  describe  only  the  more 
important  ones  now  in  use.  Each  investigator  changes  them 
according  to  his  taste. 

FLUID  MEDIA 

Bouillon  (According  to  Lb  filer). — A  cooked  infusion  of 
beef  made  slightly  alkaline  with  sodium  carbonate :  500  grams 
of  finely  chopped  raw  lean  beef  is  placed  in  a  wide-mouthed 
jar  and  covered  with  i  liter  of  water;  this  is  left  standing  twelve 
hours  with  occasional  shaking.  It  is  then  strained  through 
cheese-cloth,  the  white  meat  remaining  being  pressed  until  one 
liter  of  the  blood-red  meat-water  has  been  obtained.  The 
meat-water  must  now  be  cooked,  but  before  doing  this,  in 
order  to  prevent  all  the  albumin  from  coagulating,  10  parts  of 
peptone  powder  and  5  parts  of  common  salt  are  added  to  every 
1000  parts  meat-water.  For  water  analysis  the  salt  must  be 
omitted.  It  is  next  placed  in  the  steam-chest  or  water-bath 
for  three-quarters  of  an  hour. 

Neutralization. — The  majority  of  bacteria  grow  best  on  a 
neutral  or  slightly  alkaline  soil,  and  the  bouillon,  as  well  as 
4 


50  ESSENTIALS    OF    BACTERIOLOGY 

other  media,  must  be  carefully  neutralized  with  a  saturated 
solution  of  sodium  carbonate.  Since  too  much  alkalinity  is 
nearly  as  bad  as  none  at  all,  the  soda  must  be  added  drop  by 
drop  until  red  litmus-paper  commences  to  turn  blue.  The 
bouillon  is  then  cooked  another  hour,  and  filtered  when  cold. 
The  liquid  thus  obtained  must  be  clearly  alkaline,  and  not 
clouded  by  further  cooking.  If  cloudiness  occur,  the  white 
of  an  egg  and  further  boiling  will  clear  the  same.  To  make 
bouillon,  beef-extract  can  be  used  instead  of  fresh  meat,  2  grams 
to  i  liter  of  water.  This  is  boiled  with  5  grams  of  salt  and  10 
of  peptone,  neutralized  as  above,  and  filtered  when  cold. 

Schultz's  Method  of  Neutralization. — A  more  accurate 
method  of  obtaining  the  required  reaction  is  to  use  an  alcoholic 
solution  (J  per  cent.)  of  phenolphthalein  as  an  indicator;  a 
few  drops  of  this  are  mixed  with  10  c.c.  of  the  bouillon  and 
40  c.c.  of  water,  and  heated  to  boiling  and  while  hot,  ^  normal 
sodium  hydroxid  from  a  buret  is  added,  drop  by  drop,  until  a 
faint  pink  color  appears.  An  average  is  taken  from  three 
different  samples,  and  the  amount  of  soda  needed  for  the 
entire  quantity  of  bouillon  is  calculated  therefrom,  and  is 
added  in  the  form  of  normal  soda  solution.  The  reaction 
is  then  expressed  by  the  +  sign  if  acid  and  the  —  sign  if 
alkaline. 

A  bouillon  that  reacts  neutral  to  litmus  is,  on  an  average, 
+  25  to  phenolphthalein,  i.  e.,  requires  25  c.c.  of  normal 
soda  solution  to  a  liter  to  make  it  neutral  to  the  latter  indi- 
cator. 

An  optimum  reaction  is  one  about  midway  between  the 
neutral  point  of  litmus  and  the  neutral  point  of  phenol- 
phthalein. 

Glucose  broth,  which  is  a  good  medium  for  anaerobic  organ- 
isms, consists  of  bouillon  to  which  i  to  2  per  cent,  of  grape- 
sugar  has  been  added.  Glycerin  broth  is  bouillon  to  which  6  to 
8  per  cent,  of  glycerin  has  been  added  after  filtration. 

Sterilization  of  the  Bouillon.— Erlenmeyer  flasks  (little 
conic  glass  bottles)  or  test-tubes  plugged  and  properly  sterilized 
are  filled  one-third  full  with  the  bouillon,  and  placed  with 


NUTRIENT   MEDIA  51 

their  contents  in  the  steam-chest.  They  are  left  in  steam  of 
100°  C.  fifteen  minutes  for  three  successive  days,  after  which 
the  tubes  and  bouillon  are  ready  for  use. 

Solid  Media. — The  knowledge  of  bacteria  and  germs  or 
molds  settling  and  growing  upon  slices  of  potato  exposed  to 
the  air  led  to  the  use  of  solid  media  for  the  artificial  culture  of 
the  same.  It  was  thus  learned  that  each  germ  tends  to  form 
a  separate  colony  and  remain  isolated. 

Potato -cultures. — A  ripe  potato  with  a  smooth  skin  is  the 
best. 

Several  are  brushed  and  scrubbed  with  water  to  get  rid  of  the 
dirt,  and  the  ''eyes"  are  cut  out. 

Next  placed  in  i  :  500  solution  of  bichlorid  of  mercury  for 
one-half  hour.  Then  in  the  steam-chest  for  three-quarters 
of  an  hour. 

In  the  mean  time  a  receptacle  is  prepared  for  them.  This 
is  called  the  moist  chamber. 


Fig.  20. — Moist  chamber  for  potatoes. 

The  moist  chamber  consists  of  two  large  shallow  dishes,  one, 
the  larger,  as  a  cover  to  the  other  (Fig.  20) . 

These  dishes  are  washed  in  warm  distilled  water. 

A  layer  of  filter-paper  moistened  with  15  to  30  drops  of  a 
i  :  1000  bichlorid  solution  is  placed  in  the  bottom  of  the  glass 
dish. 

The  operator  now  prepares  his  own  hands,  rolling  up  his 
coat-sleeves  and  carefully  washing  his  hands,  then  taking  a 
potato  from  the  steam-oven  and,  holding  it  between  his  thumb 
and  index-finger  in  the  short  axis,  he  divides  the  potato  in  its 


ESSENTIALS    OF    BACTERIOLOGY 


long  axis  with  a  knife  that  has  been  passed  through  the  flame. 
The  two  halves  are  kept  in  contact  until  they  are  lowered  into 
the  moist  chamber,  when  they,  of  their  own  weight,  fall  aside, 
the  cut  surface  uppermost.  They  are  then  ready  for  inocula- 
tion. 

Esmarch's  Cubes.— The  potato  is  first  well  cleaned  and 
peeled.  It  is  then  cut  in  cubes  \  inch  in  size. 

These  are  placed,  each  in  a  little  glass  dish  or  tray,  and  then 
in  steam-chest  for  one-half  hour,  after  which  they  are  ready 
for  inoculation  (the  dishes  first  having  been  sterilized  in  hot-air 
oven) . 

Test-tube  Potatoes. — Cones  are  cut  out  of  the  peeled 
potato  and  placed  in  test-tubes,  which  can  then  be  plugged 
and  easily  preserved. 

Roux's  test-tube  (Fig.  21),  specially  designed 
for  potato  cultures,  consists  of  a  tube  with  a 
small  constricted  portion  at  the  bottom,  in  which 
water  may  be  kept  to  keep  the  potato  moist. 

Manner  of  Inoculation. — With  a  platinum 
rod  or  a  spatula  (sterilized)  the  material  is  spread 
upon  one  of  the  slices,  keeping  free  of  the 
edges.  The  growth  on  this  first,  or  original, 
potato  will  be  quite  luxuriant,  and  the  individual 
colonies  often  difficult  to  recognize;  therefore 
dilutions  are  made  (Fig.  22). 

From  the  original  or  first  slice  a  small  portion, 
including  some  of  the  meat  of  the  potato,  is 
spread  upon  the  surface  of  a  second  slice, 
which  is  first  dilution.  From  this  likewise  a 
small  bit  is  taken  and  spread  on  a  third  slice,  or 
second  'dilution,  and  here  usually  the  colonies 
will  be  sparsely  enough  settled  to  study  them  in  their  indi- 
viduality. 

This  is  the  principle  carried  on  in  all  the  cultivations.  It 
is  a  physical  analysis. 

Potato  and  Bread  Mash. — These  pastes  are  used  chiefly  in 
the  culture  of  molds  and  yeasts.  Peeled  potatoes  are  mashed 


Fig.  21. — Tube 
for  potato  cul- 
ture. 


SOLID  TRANSPARENT  MEDIA  53 

with  distilled  water  until  thick,  and  then  sterilized  in  flasks 
three-quarters  of  an  hour  for  three  successive  days. 


Fig.  22. — Method  of  inoculation  (Woodhead  and  Hare). 

Bread  Mash. — Bread  devoid  of  crust,  dried  in  an  oven,  and 
then  pulverized  and  mixed  with  water  until  thick,  and  sterilized 
as  above. 


CHAPTER  IX 
SOLID  TRANSPARENT  MEDIA 

Solid  transparent  media  are  prepared  from  materials  which 
can  be  used  for  microscopic  purposes  and  which  can  readily  be 
converted  into  liquids.  Such  are  the  gelatin  and  agar  culture- 
media. 

Gelatin. — Gelatin  is  obtained  from  bones  and  tendons,  and 
consists  chiefly  of  chondrin  and  gluten. 

The  French  Golden  Medal  brand  is  the  one  most  in  use, 
found  in  long  leaves  with  ribbed  lines  crossing  them. 

Koch-Lbffler  10  per  cent.  Bouillon-gelatin.— To  the 
meat-water  as  made  for  the  bouillon  are  added  100  grams 
gelatin,  10  grams  peptone,  5  grams  salt,  to  each  1000  grams 


54 


ESSENTIALS    OF    BACTERIOLOGY 


of  the  meat- water;  or  to  the  bouillon  made  from  beef-extract 

the  gelatin  is  added;  this  is  placed  in  a  flask  and  gently  heated 

until  the  gelatin  is  dissolved. 

It  is  neutralized  with  the  soda  and  then  cooked  in  water-bath 

or  carefully  boiled  over  flame  for  one  hour  or  more  until  the 

liquid  seems  clear,  then  add  white  of  an  egg  and  boil  one- 
quarter  of  an  hour  longer; 
the  egg  will  produce  a  clearer 
solution  and  save  much 
trouble.  A  small  portion, 
while  hot,  is  now  filtered 
into  a  test-tube  and  tested 
for  alkalinity,  and  then  re- 
heated several  times,  watch- 
ing if  a  cloudy  precipitate 
forms. 

If  the  fluid  remains  clear 
upon  cooling,  the  remainder 
of  the  material  can  be  fil- 
tered. It  must  be  accom- 
plished while  hot,  else  the 
gelatin  will  coagulate  and 
prevent  further  filtration. 
This  can  be  carried  on 
either  by  keeping  hot  the  so- 
lution continually  in  water- 
bath,  and  only  filtering  a 
small  quantity  at  a  time 
through  the  filter,  or  keep- 
ing the  filter  itself  hot,  either 

with   a    hot-water   filter  or  placing  the  filter  in  steam-chest 

(Fig-  23). 
Clouding  of  Gelatin. — If  the  gelatin  does  not  come  out  clear 

or  becomes  turbid  on  cooling,  it  may  be  due  to  several  things — 

1.  The  filter-paper  too  thin  or  impure. 

2.  Too  strongly  alkaline. 

3.  Cooked  too  long  or  not  long  enough. 


Fig.   23. — Hot-water  filter. 


SOLID   TRANSPARENT   MEDIA  55 

The  addition  of  the  white  of  an  egg,  as  before  mentioned, 
will  often  clear  it  up ;  if  this  avails  not,  refiltering  several  times 
and  attention  to  the  few  points  mentioned  will  produce  a  clear 
solution. 

Sterilizing  the  Gelatin.— The  gelatin  is  kept  in  little  flasks 
or  poured  at  once  into  sterile  test-tubes,  careful  not  to  wet  the 
neck  where  the  cotton  enters,  lest  when  cool  the  cotton  plug 
stick  to  the  tube. 

The  tubes  are  then  placed  in  steam-chest  for  three  successive 
days,  fifteen  minutes  each  day  (or  in  water-bath  one  hour  a 
day  for  three  days).  Then  set  aside  in  a  temperature  of  15° 
to  20°  C.,  and  if  no  germs  develop  and  the  gelatin  remains 
clear,  it  can  be  used  for  cultivation  purposes. 

Modification. — The  amount  of  gelatin  added  to  the  meat- 
water  can  be  variously  altered,  and  instead  of  making  gelatin 
bouillon,  milk,  blood,  serum,  urine,  and  agar  can  be  added. 
Glycerin  (4  to  6  per  cent.)  is  a  common  addition,  and  sometimes 
reducing  agents  to  absorb  the  oxygen  are  mixed  with  it. 

Agar-agar. — This  agent,  which  is  of  vegetable  origin,  derived 
from  sea-plants  gathered  on  the  coasts  of  India  and  Japan,  has 
many  of  the  properties  of  gelatin,  retaining  its  solidity  at  a 
much  higher  temperature;  it  becomes  liquid  at  90°  C.  and  con- 
geals again  at  45°  C.  Gelatin  will  liquefy  at  35°  C. 

Agar  is  not  affected  very  much  by  the  peptonizing  action  of 
the  bacteria — 38°  C.  is  the  temperature  at  which  most  patho- 
genic germs  grow  best. 

Preparation  of  Agar-agar  Bouillon  or  Nutrient  Agar. — 
The  ordinary  bouillon  is  first  made,  and  then  the  agar  cut  in 
small  pieces,  added  to  the  bouillon  (15  grams  of  agar  to  1000 
grams  of  bouillon) .  It  is  allowed  to  stand  several  minutes  until 
the  agar  swells,  and  then  placed  in  water-bath  or  steam-chest 
for  six  hours  or  more.  It  is  then  neutralized,  very  little  of  the 
alkaii  being  sufficient. 

A  white  of  an  egg  added,  and  boiled  for  several  hours  longer, 
when,  even  if  not  perfectly  clear,  it  is  filtered. 

The  filtering  process,  very  difficult  because  of  the  readiness 
with  which  the  agar  solidifies,  must  be  done  in  steam-chest  or 


56  ESSENTIALS    OF    BACTERIOLOGY 

with  hot-water  filter,  and  very  small  quantities  passed  through 
at  a  time,  changing  the  filter-paper  often. 

Cotton  can  be  used  instead  of  filter-paper,  or  filtering  entirely 
dispensed  with,  simply  decanting. 

As  agar  is  seldom  clear,  a  little  more  or  less  opaqueness  is 
permissible.  The  test-tubes  are  filled  as  with  the  gelatin,  and 
sterilized  in  the  same  manner.  While  cooling,  some  of  the 
tubes  can  be  placed  in  a  slanting  position,  so  as  to  obtain  a  larger 
surface  to  work  upon. 

Water  of  condensation  will  usually  separate  and  settle  at  the 
bottom,  or  a  little  white  sediment  remain  encysted  in  the  center; 
this  cannot  easily  be  avoided,  nor  does  it  form  any  serious 
obstacle. 

The  crude  agar  should  first  be  rinsed  in  water,  and  then  in 
5  per  cent,  acetic  acid  and  clear  water  again,  to  rid  it  of  impuri- 
ties. If  agar  is  boiled  thoroughly  over  a  hot  flame  or  in  an 
autoclave,  it  can  be  filtered  much  more  readily.  The  main 
point  is  to  see  that  all  the  agar  is  dissolved. 

It  has  been  suggested  to  pour  the  hot  agar  into  high  cylin- 
dric  glass  vessels  and  allow  it  to  cool  slowly  in  the  steam-oven, 
the  flame  having  been  gradually  lowered  and  then  turned 
out.  After  a  time  the  cloudy  portion  will  form  a  sediment  at 
the  bottom;  the  agar  can  then  be  shaken  out  as  a  long  cylinder 
and  the  cloudy  portion  cut  off. 

The  Japanese  Method. — Yokote  prepares  agar  as  follows:  the 
meat  is  cooked  in  water  over  a  sand-bath  one  and  one -half 
hours.  Filtered,  chopped  agar  is  then  added,  and  the  mixture 
cooked  one  hour  longer;  peptone  and  salt  added  next.  Neutral- 
ization. After  the  mixture  has  cooled  to  about  50°  C.,  whites 
of  two  eggs  are  added  and  the  mixture  shaken  thoroughly. 

Again  the  mixture  is  placed  on  the  sand-bath  and  heated  to 
110°  C.  and  over  for  one  and  one-half  to  two  hours,  and  then 
filtered  through  ordinary  filter-paper.  Yokote  claims  that 
by  this  procedure  the  agar  can  be  filtered  as  easily  as  bouillon 
and  without  any  loss.  (Water  must  be  added  before  filtering 
to  supply  loss  from  evaporation.) 

The  agar  may  be  boiled  separately  in  some  of  the  water 


SOLID   TRANSPARENT   MEDIA 


57 


ordinarily  used  in  the  meat-water,  and  then,  when  well  dis- 
solved, the  concentrated  meat-water  infusion  is  added  and  the 
two  solutions  boiled  together. 

Glycerin-agar. — The  addition  of  4  to  6  per  cent,  of  glycerin 
to  nutrient  agar  greatly  enhances  its  value  as  a  culture-medium. 

Gelatin-agar. — A  mixture  of  5  per  cent,  gelatin  and  0.75 
per  cent,  agar  combines  in  it  some  of  the  virtues  of  both  agents. 

Blood-serum. — Blood-serum,  being  rich  in  albumin,  coagu- 
lates very  easily  at  70°  C.,  and  if  this  temperature  is  not 
exceeded,  a  transparent,  solid  substance  is  obtained  upon  which 
the  majority  of  bacteria  develop,  and  some  with  preference. 

Preparation  of  Nutrient  Blood -serum. — If  the  slaughter 
of  the  animal  can  be  supervised,  it  were  best  to  have  the  site  of 
the  wound  and  the  knife  sterilized,  and  sterile  flasks  (Fig.  24)  at 
hand  to  receive  the  blood  directly  as  it  flows. 

The  blood  is  placed  on  ice  forty-eight 
hours,  and  the  serum  is  drawn  out  with 
sterile  pipets  into  test-tubes;  these  are  placed 
obliquely  in  an  oven  where  the  temperature 
can  be  controlled  and  maintained  at  a  cer- 
tain degree.  (See  Fig.  25.) 

Incubators  or  Brood-ovens. — Incubators 
or  brood-ovens  consist  essentially  of  a  double- 
walled  zinc  or  copper  chest,  the  space  between 
the  walls  being  filled  with  water. 

The  oven  is  covered  with  some  imperme- 
able material  to  prevent  the  action  of  the 
surrounding  atmosphere  (Fig.  26).  It  is 
supplied  with  a  thermometer  and  a  regu- 
lator. The  regulator  is  connected  with  a 
Bunsen  burner,  and  keeps  the  temperature 
at  a  certain  height. 

There  are  several  forms  of  regulators  in  use,  and  new  ones 
are  invented  continually.  The  size  of  the  flame  in  some  is 
regulated  by  the  expansion  of  mercury,  which,  as  it  rises, 
lessens  the  opening  of  the  gas-supply.  The  mercury  con- 
tracting on  cooling  allows  more  gas  to  enter  again  (Fig.  27). 


Fig.  24.— Flask 
to  receive  blood- 
serum. 


58  ESSENTIALS    OF    BACTERIOLOGY 

Koch  has  invented  a  safety  burner  by  which  the  gas  supply  is 
shut  off  should  the  flame  accidentally  go  out. 

Coagulation  of  Blood-serum. — The  tubes  of  blood-serum 
having  been  placed  in  the  oven,  are  kept  at  a  temperature 
of  65°  to  68°  C.  until  coagulation  occurs;  then  removed  and 
sterilized. 


Fig.  25. — Thermostat  for  blood -serum. 

Sterilization  of  Blood-serum. — The  tubes  are  placed  three 
to  four  days  in  incubation  at  58°  C.,  and  those  tubes  which 
show  any  evidences  of  organic  growth  are  discarded. 

If,  now,  at  the  end  of  a  week,  the  serum  remains  sterile  at  the 
ordinary  temperature  of  the  room,  it  can  be  used  for  experi- 
mental purposes. 

Perfectly  prepared  blood-serum  is  transparent,  of  a  gelatin- 
like  consistence,  and  straw  color.  It  will  not  liquefy  by  heat, 
though  bacteria  can  digest  it.  Water  of  condensation  always 
forms,  which  prevents  the  drying  of  the  serum.  Blood-serum 


SOLID   TRANSPARENT   MEDIA  59 

may  be  prepared  in  a  shorter  way  by  coagulating  the  serum  at 
a  temperature  short  of  boiling-point.  Sterilization  is  completed 
in  three  days  by  exposing  the  tubes  to  a  temperature  of  about 
90°  C.  each  day  for  five  minutes.  Tubes  so  prepared  are 
opaque  and  white. 

Preservation    of  Blood-serum    in    Liquid    State. — Kirchner 
advises  the  use  of  chloroform.     To  a  quantity  of  serum  in  a 


Fig.  26. — Incubator.  Fig.   27. — Thermoregulators. 

well-stoppered  flask  a  small  amount  of  chloroform  is  added— 
enough  to  form  about  a  2  mm.  layer  on  the  bottom.  If  the 
chloroform  is  not  allowed  to  evaporate,  the  serum  remains 
sterile  for  a  long  time.  When  needed  for  use,  test-tubes  are 
filled  and  placed  in  a  water-bath  at  50°  C.  until  all  chloroform 
has  been  driven  off  (determined  by  absence  of  characteristic 
odor);  the  serum  is  then  solidified  and  sterilized  as  in  the 
ordinary  way. 


60  ESSENTIALS    OF    BACTERIOLOGY 

Human  blood-serum  derived  from  placenta,  serum  from 
ascitic  fluid  and  ovarian  cysts,  is  prepared  in  a  similar 
manner  to  the  above. 

Blood  coagulum,  suggested  by  the  author,  is  the  blood  itself 
(not  the  serum  only)  coagulated  in  test-tubes.  It  is  dark  brown 
in  color  and  allows  some  colonies  of  bacteria  to  be  more  visible. 
It  requires  less  time  to  prepare,  and  is  not  so  likely  to  become 
contaminated  as  when  the  serum  is  used. 

L6 filer's  Blood-serum  Mixture. — See  p.  124. 

Peptone  Solution  (Dunham's) . — Sodium  chlorid,  0.5  parts; 
peptone,  i  part;  water,  100  parts.  Boil,  filter,  and  sterilize. 
Useful  to  detect  presence  of  indol. 

Other  Nutrient  Media. — Milk,  urine,  decoctions  of  various 
fruits  and  plants,  and  for  cultivating  anaerobic  bacteria,  eggs. 

Lactose  (or  Dextrose)  Litmus-agar  (for  Water  Bacteria). 
—To  nutrient  agar,  i  per  cent,  of  dextrose  or  lactose  is  added 
just  before  sterilization.  The  reaction  should  be  neutral  to 
phenolphthalein.  Then,  if  the  medium  is  to  be  used  in  tubes, 
sterilized  azolitmin,  i  per  cent,  (aqueous  solution)  is  added 
just  before  the  final  sterilization.  If  Petri  dishes  are  used, 
the  azolitmin  solution  is  not  added  until  the  medium  is  ready 
to  be  poured. 

Blood-agar. — Human  or  other  blood  is  obtained  direct 
from  the  body  under  strict  aseptic  conditions,  and  a  few  drops 
smeared  over  the  surface  of  agar  in  tubes  or  plates.  These  are 
then  placed  in  the  incubator  for  a  few  days,  and  the  contami- 
nated ones  are  rejected.  This  media  is  used  for  influenza  bacilli 
and  gonococci. 

Dunham's  rosalic  acid  solution  consists  of  the  following: 

Peptone  solution  (Dunham) 100  c.c. 

2  per  cent,  solution  rosalic  acid 0.5  gm. 

Alcohol  (80  per  cent.) 100  c.c.— M. 

To  detect  acids  and  alkalis. 

Eisner's  Medium  (for  Typhoid)  (Potassium  lodid— 
Potato -gelatin). — Five  hundred"  grams  of  peeled  and  washed 


SOLID   TRANSPARENT   MEDIA  6 1 

potatoes  are  mashed  and  pressed  through  a  fine  cloth.  The 
juice  is  allowed  to  settle,  is  filtered,  and  after  one  hour's  cook- 
ing has  added  to  it  10  per  cent,  gelatin;  then  2\  c.c.  yL  normal 
sodium  hydroxid  solution,  and  finally  i  per  cent,  potassium 
iodid. 

Typhoid  Medium  of  Hiss. — This  consists  of  a  slightly  acid 
mixture  of  gelatin  and  agar,  beef-extract,  sodium  chlorid,  and 
dextrose,  used  in  different  proportions  for  plate  and  tube  cul- 
tures. It  is  semisolid  in  character,  and  facilitates  the  identi- 
fication of  the  motile  typhoid  bacilli,  which  produce  a  uniform 
clouding  through  the  medium  in  tubes. 

Bile-salt  Media  (MacConkey). — Used  for  intestinal  bac- 
teria; stock  solution  consists  of: 

Sodium  taurocholate 0.5  gm. 

Witte's  peptone 2.0 

Distilled  water 100.0  c.c. — M. 

To  which  is  added  as  an  indicator,  neutral  red  (crimson 
with  acid,  yellow  with  an  alkali)  in  i  per  cent,  solution,  0.5  c.c., 
and  dextrose  or  lactose.  The  fluid  is  placed  in  fermentation 
tubes  and  sterilized  for  ten  minutes  on  three  successive  days. 

Agar  can  be  added  to  make  bile-salt  lactose  agar.  Bacillus 
coli  and  Bacillus  typhosus  grow  readily  on  this  media;  other 
water  bacteria  are  inhibited,  especially  at  40°  C.;  acid-formers 
and  gas-formers  are  denoted  by  rose-red-colored  colonies. 

Milk  Culture-medium. — The  milk  used  should  be  fresh 
and  should  be  placed  on  ice  for  eight  to  ten  hours  to  allow  the 
cream  to  rise;  the  skimmed  milk  is  siphoned  off  into  flasks 
or  tubes  and  sterilized  for  three  successive  days.  Litmus  is 
often  added,  or  sterile  i  per  cent,  azolitmin  solution. 

Urine  Media  (for  Gonococci) : 

Urine  (sterile  taken) i  part. 

2  per  cent,  agar  solution i 

Fresh  Egg  Cultures  (After  Hueppe).— The  eggs  in  the 
shell  are  carefully  cleaned,  washed  with  sublimate,  and  dried 
with  cotton. 


62 


ESSENTIALS    OF    BACTERIOLOGY 


The  inoculation  occurs  through  a  very  fine  opening  made  in 
the  shell  with  a  hot  platinum  needle;  after  inoculation,  the  open- 
ing is  covered  with  a  piece  of  sterilized 
paper,  and  collodion. 

Boiled  Eggs.— Eggs  boiled,  shell  re- 
moved over  small  portion,  and  the  co- 
agulated albumen  stroked  with  the 
material. 

Guinea-pig  Bouillon. — The  flesh  of 
guinea-pigs,  as  well  as  that  of  other 
experiment  animals,  is  used  instead  of 
beef  in  the  preparation  of  bouillon,  for 
the  growth  of  special  germs. 

The  extracts  of  different  organs  have 
been  added  to  the  various  media  for 
experimentation. 

Fermentation  Tube. — For  showing 
the  presence  of  gas  or  fermentation  the 
Smith  tube  (Fig.  28)  or  some  of  its 
modifications  must  be  used.  The  closed 
end  and  part  of  the  bulb  are  filled  with 
the  glucose  or  dextrose  bouillon  and 

sterilized  at  low  temperatures  for  three  successive  days,  then 
inoculated,  and  placed  in  the  incubator.  Gas  forms  gradu- 
ally, displacing  the  fluid  in  the  closed  end. 


Fig.     28.— Smith's    fer- 
mentation tube. 


CHAPTER  X 
INOCULATION  OF  GELATIN  AND  AGAR 

Glass  Slide  Cultures. — Formerly  the  gelatin  was  poured  on 
little  glass  slides,  such  as  are  used  for  microscopic  purposes, 
and  after  it  had  become  hard,  inoculated  in  separate  spots  as 
with  potatoes. 


INOCULATION   OF   GELATIN  AND   AGAR  63 

Test-tube  Cultures. — The  gelatin,  agar,  or  blood-serum 
having  solidified  in  an  oblique  position,  is  smeared  on  the  surface 
with  the  material,  and  the  growth  occurs,  or  the  medium  is 
punctured  with  a  stab  of  the  platinum  rod  containing  the 
material.  The  former  is  called  a  stroke  or  smear  culture,  the  latter 


Fig.  29. — Manner  of  holding  tubes  for  inoculation:  a,  Tube  with  material; 
b,  tube  to  be  inoculated;  c,  cotton  plugs  (after  Woodhead  and  Hare). 

a  stab  or  thrust  culture.  In  removing  the  cotton  plugs  from 
the  sterile  tubes  to  carry  out  the  inoculation,  the  plugs  should 
remain  between  the  fingers  in  such  a  way  that  the  part  which 
comes  in  contact  with  the  mouth  of  the  tube  will  not  touch 
anything  (Fig.  30). 


64  ESSENTIALS    OF    BACTERIOLOGY 

It  is  well  to  pass  the  mouth  of  the  tube  and  the  cotton  plugs 
through  a  flame,  scorching  the  latter  before  reinserting. 

After  the  needle  has  been  withdrawn,  the  plugs  are  reinserted 
and  the  tubes  labeled  with  the  kind  and  date  of  culture. 

Plate  Cultures. — This  method  once  common  is  now  seldom 
or  ever  used,  and  has  been  superseded  by  the  Petri  dishes;  -as 
a  matter  of  history  the  description  is  retained.  Several  tubes 
of  the*  culture-medium  are  made  liquid  by  heating  in  water- 
bath,  and  then  inoculated  with  the  material  as  follows.  A 
looped  platinum  needle  is  dipped  into  the  material  and  then 
shaken  in  the  tube  of  liquid  media  (gelatin,  agar,  etc.). 

This  first  tube  is  called  original.  From  this  three  drops 
(taken  with  the  looped  platinum  rod)  are  placed  in  a  second 


Fig.  30. — Manner  of  holding  plugs. 

tube,  the  rod  being  shaken  somewhat  in  the  gelatin  or 
agar;  this  is  labeled  first  dilution  (a  colored  pencil  is  useful 
for  such  markings) .  From  the  first  dilution  three  drops  are 
taken  into  a  third  tube,  which  becomes  the  second  dilution 
(Fig.  29). 

The  plugs  of  cotton  must  be  replaced  after  each  inoculation, 
and  during  the  same  must  be  carefully  protected  from  contami- 
nation. 

Glass  Plates. — The  larger  the  surface  over  which  the  nutrient 
medium  is  spread,  the  .more  isolated  will  the  colonies  be; 
window  glass  cut  in  rectangular  plates  6x4  inches  in  size  is 
used;  about  ten  such  plates  are  cleaned  with  dry  towel  and 
placed  in  a  small  iron  box  or  wrapped  in  paper,  and  steril- 


INOCULATION   OF   GELATIN   AND    AGAR 


ized  in  the  hot-air  oven  at  a  temperature  of  150°  C.  for  ten 
minutes  (Fig.  31).     When  the  plates  have  cooled,  they  are 
placed  upon  an  apparatus  designed  to  cool  and  solidify  the 
liquid  media,  which  is  now  poured  upon 
the  plates  from  the  inoculated  test-tubes. 

Nivellier    Leveling    and   Cooling  Ap 
paratus. — Ice  and  water  are  placed  in  a 
shallow  round  glass  tray;  on  top  of  this  a 
square    plate  of  glass,   upon   which 
culture-plate  is  placed,  and  covering  this, 
a  bell-glass. 

The  whole  is  upon  a  low  wooden 
tripod,  the  feet  of  which  can  be  raised 
or  lowered,  and  a  little  spirit-level  used 
to  adjust  it  (Fig.  32).  The  glass  plate 


a 

a 
ic 

s, 

n 

r\ 

A 
/ 

Fig.  31. — Iron  box  for 
glass  plates. 


taken  out  of  the  iron  box  is  placed  under  the  bell-glass.  The 
tube  containing  the  gelatin  is  held  in  the  flame  a  second  to 
singe  the  cotton  plug  to  free  it  from  dust,  and  the  plug 
removed,  the  edges  of  the  tube  again  flamed,  the  bell-glass 


Fig.  32. — Nivellier  leveling  and  cooling  apparatus. 

lifted,  and  the  inoculated  gelatin  carefully  poured  on  the 
plate,  leaving  about  one-third  inch  margin  from  the  borders; 
the  lips  of  the  tube  being  sterile,  can  be  used  to  spread  the  media 
evenly.  If  the  plate  is  at  all  cool,  the  fluid  will  solidfy  as  it  is 
being  spread.  The  glass  cover  is  replaced  until  the  gelatin  or 
agar  is  quite  solid,  to  prevent  contamination. 


66 


ESSENTIALS    OF    BACTERIOLOGY 


When  the  gelatin  is  congealed,  the  plate  is  placed  upon  a 
little  glass  bench  or  stand  in  the  moist  chamber. 

The  Moist  Chamber. — Prepared  out  of  two  glass  dishes,  as 
for  the  potato-cultures.  The  glass  benches  are  so  arranged  that 
one  stands  upon  the  other  (Fig.  33).  In  order  to  avoid  con- 
fusion, a  slip  of  paper  with  a  number  written  on  it  is  placed 


Fig-  33- — Moist  chamber  with  plates  on  benches. 

on  the  bench  beneath  each  plate.  As  the  original  or  first  plate 
would  have  the  colonies  developed  in  greatest  profusion,  it  is 
placed  the  first  day  on  the  topmost  bench;  but,  since  the  colonies 
would  be  likely  to  overrun  the  plate  and  allow  the  gelatin  to 
drop  on  the  lower  plates,  it  is  best,  as  soon  as  evidences  of  growth 
appear,  to  place  it  below,  and  watch  the  third  plate  or  second 


Fig.  34. — Petri  dish  for  making  plate  cultures. 

dilution  for  the  characteristic  colonies,  forgetting  not  all  this 
time  to  change  the  numbers  accordingly. 

The  date  of  culture  and  the  name  can  be  written  upon  the 
moist  chamber. 

Petri  Saucers. — Agar  hardens  very  quickly,  even  without 
any  especial  means  for  cooling,  and  it  does  not  adhere  very 
well  to  the  glass.  Therefore  it  is  better  to  follow  the  method 


INOCULATION   OF   GELATIN   AND   AGAR  67 

of  Petri  and  use  little  shallow  glass  dishes,  one  covering  the 
other  (Fig.  34) .  They  are  first  sterilized  by  dry  heat,  and  then 
the  inoculated  gelatin  or  agar  is  poured  into  the  lower  dish, 
covered  by  the  larger  one,  and  placed  in  some  cool  place, 
different  saucers  being  used  for  each  dilution. 

This  method  is  very  useful  for  transportation,  and  does  away 
with  the  cooling  apparatus  and  moist  chamber;  the  saucers 
can  be  viewed  under  microscope  similar  to  the  glass  plates,  and 
have  entirely  superseded  them. 

Esmarch's  Tubes  or  Rolled  Cultures.— This  method, 
especially  used  in  the  culture  of  anaerobic  germs,  consists  in 
spreading  the  inoculated  gelatin  upon  the  inner  walls  of  the 
test-tube  in  which  it  is  contained  and  allowing  it  to  congeal. 
The  colonies  then  develop  upon  the  sides  of  the  tube  without 
the  aid  of  other  apparatus.  The  method  is  useful  whenever  a 
very  quick  and  easy  way  is  required.  The  rolling  of  the  tube 
is  done  under  ice-water  or  running  water  from  the  faucet.  The 
tube  is  held  a  little  slanting,  so  as  to  avoid  getting  too  much 
gelatin  around  the  cotton  plug. 

The  tubes  can  be  placed  directly  under  the  microscope  for 
further  examination  of  the  colonies. 

Animals  as  Culture  Media. — It  is  almost  impossible  to 
separate  certain  organisms,  such  as  the  tubercle  bacillus  and 
pneumococcus,  from  mixed  cultures  by  ordinary  plate  methods, 
and  the  plan  of  producing  the  disease  in  animals  by  inoculation, 
and  then  obtaining  the  organism  in  pure  culture,  has  to  be 
employed. 

Pure  Cultures  by  Boiling. — Spored  organisms  may  be 
separated  from  others  by  boiling  the  mixture  for  a  few 
minutes,  when  all  the  non-spored  forms  will  perish,  and  only 
the  spores  remain  to  germinate  subsequently. 


68  ESSENTIALS    OF    BACTERIOLOGY 

CHAPTER  XI 
THE  GROWTH  AND  APPEARANCES  OF  COLONIES 

Macroscopic. — Depending  greatly  upon  the  temperature 
which  should  be  about  65°  F.  (20°  C.)  for  gelatin,  and  40°  C. 
for  agar,  the  colonies  ordinarily  develop  so  as  to  be  visible  to 
the  naked  eye  in  two  to  four  days.  Some  require  ten  to  four- 
teen days,  and  others  grow  rapidly,  covering  the  third  dilution 
in  thirty-six  hours.  The  plate  should  be  looked  at  each  day. 

The  colonies  present  various  appearances  from  that  of  a 
small  dot,  like  a  fly-speck,  to  that  resembling  a  small  leaf. 


Fig-  35- — Staphylococcus  pyogenes  aureus:  colony  two  days  old,  seen  upon 
an  agar-agar  plate  (x  40)  (Heim). 

f 

Some  are  elevated,  some  depressed,  and  some,  like  cholera,  cup- 
shaped — umbilicated. 

Then  they  are  variously  pigmented.  Some  liquefy  the  gela- 
tin speedily,  others  not  at  all.  The  appearances  of  a  few  are 
so  characteristic  as  to  be  recognized  at  a  glance. 

Microscopic. — We  use  a  low-power  lens,  with  the  Abbe 
nearly  shut  out — that  is  the  narrowest  blender.  The  stage  of 


THE  GROWTH   AND  APPEARANCES  OF  COLONIES       69 

the  microscope  should  be  of  such  size  as  to  carry  a  culture 
plate  easily  upon  it. 

The  second  dilution  or  third  plate  is  usually  made  use  of— 
that  one  containing  the  colonies  sufficiently  isolated. 

These  isolated  ones  should  be  sought  for,  and  their  appear- 
ance well  noticed. 

There  may  be  two  or  three  forms  from  the  same  germ,  the 
difference  due  to  the  greater  or  less  amount  of  oxygen  that  they 
have  received,  or  the  greater  or  less  amount  of  space  that  they 
have  had  to  develop  in. 

The  microscopic  picture  varies  greatly;  now  it  is  like  the 
gnarled  roots  of  a  tree,  and  now  like  bits  of  frosted  glass;  the 


Fig.  36. — Microscopic  appear-  Fig.  37. — Klatsch  preparations, 

ances  of  colonies 

pictures  are  very  characteristic,  and  the  majority  of  bacteria 
can  be  told  thereby  (Fig.  35). 

Impression  or  "  Klatsch  "  Preparations. — In  order  more 
thoroughly  to  study  a  certain  colony  and  to  make  a  permanent 
specimen  of  the  same,  we  press  a  clean  cover-glass  upon  the 
particular  colony,  and  it  adheres  to  the  glass.  It  can  then  be 
stained  or  examined  so.  The  Germans  give  the  name  of 
"  Klatsch' '  to  such  preparations.  Many  beautiful  pictures  can 
be  so  obtained. 

Fishing. — To  obtain  and  examine  the  individual  members  of 
a  particular  colony  the  process  of  fishing,  as  it  is  called,  is 
resorted  to. 


7o 


ESSENTIALS  OF  BACTERIOLOGY 


The  colony  having  been  placed  under  the  field  of  the  micro- 
scope, a  long  platinum  needle,  the  point  slightly  bent,  is  passed 
between  the  lens  and  the  plate  so  as  to  be  visible  through  the 
microscope,  then  turned  downward  until  the  colony  is  seen  to 


Fig.  38. — Types  of  growth  in  stab-cultures:  A,  Non-liquefying:  i,  Fili- 
form (Bacillus  coli);  2,  beaded  (Streptococcus  pyogenes);  3,  echinate  (Bac- 
terium acidi  lactici);  4,  villous  (Bacterium  murisepticum);  5  arborescent 
(Bacillus  mycoides).  B,  Liquefying:  6,  Crateriform  (Bacillus  vulgare, 
twenty-four  hours);  7,  napiform  (Bacillus  subtilis,  forty-eight  hours);  8, 
infundibuliform  (Bacillus  prodigiosus);  9,  saccate  (Microsporon  Finkleri); 
10,  stratiform  (Psorospermum  fluorescens)  (Frost). 

be  disturbed,  and  the  needle  is  dipped  into  the  colony.  This 
procedure  must  be  carefully  done,  lest  a  different  colony  be 
disturbed  than  the  one  looked  at,  and  an  unknown  or  unwanted 
germ  obtained. 

After  the  needle  has  entered  the  particular  colony,  it  is  with- 


CULTIVATION   OF   ANAEROBIC   BACTERIA  71 

drawn,  and  the  material  thus  obtained  is  further  examined  by 
staining  and  animal  experimentation.  The  bacteria  are  then 
again  cultivated  by  inoculating  fresh  gelatin,  making  stab-  and 
stroke  cultures. 


Fig.  39. — Types  of  streak  cultures:  i,  Filiform  (Bacillus  coli);  ;>,  echinu- 
late  (Bacterium  acidi  lactici);  3,  beaded  (Streptococcus  pyogenes);  4,  effuse 
(Bacillus  vulgaris);  5,  arborescent  (Bacillus  mycoides)  (Frost). 

It  is  necessary  to  transfer  the  bacteria  to  fresh  media  about 
every  six  weeks,  as  the  products  of  growth  and  decay  given  off 
by  the  organisms  destroy  them.  Stroke  and  stab  test-tube 
cultures  are  more  characteristic  than  plate  cultures,  as  the 
types  in  figures  38  and  39  show. 


CHAPTER  XII 
CULTIVATION  OF  ANAEROBIC  BACTERIA 

SPECIAL  methods  are  necessary  for  the  culture  of  the  anae- 
robic variety  of  bacteria  in  order  to  procure  a  space  devoid  of 
oxygen. 

Liborius's  High  Cultures. — The  tube  is  filled  about  three- 
quarters  full  with  gelatin,  which  is  then  steamed  in  a  water- 
bath  and  allowed  to  cool  to  40°  C.,  when  it  is  inoculated  by 


72  ESSENTIALS    OF    BACTERIOLOGY 

means  of  a  long  platinum  rod  with  small  loop,  the  movement 
being  a  rotary  vertical  one,  and  the  rod  going  to  the  bottom 
of  the  tube. 

The  gelatin  is  next  quickly  solidified  under  ice;  very  little 
air  is  present.  The  anaerobic  germs  will  grow  from  the 
bottom  upward,  and  any  aerobins  present  will  develop  first 
on  top,  this  method  being  one  of  isolation. 

From  the  anaerobic  germ  grown  in  the  lower  part,  a  stab- 
culture  is  made  into  another  tube  containing  three-quarters 


Fig.  40.— Liborius's  method. 


Fig.  41. — Hesse's  method  of  making 
anaerobic  cultures  (McFarland). 


gelatin,  the  material  being  obtained  by  breaking  test-tube  with 
the  culture.  (See  Fig.  40.) 

Hesse's  Method. — A  stab-culture  having  been  made  with 
anaerobic  germs,  gelatin  in  a  semisolid  condition  is  poured 
into  the  tube  until  it  is  full,  thus  displacing  the  air  (Fig.  41). 

Esmarch's  Method.— Having  inoculated  a  tube,  the  gela- 
tin is  rolled  out  on  the  walls  of  the  tube,  a  "  roll  culture," 
and  the  rest  of  the  interior  is  filled  with  gelatin,  the  tube 


CULTIVATION   OF   ANAEROBIC   BACTERIA 


73 


being  held  in  ice-water.  The  colonies  develop  upon  the  sides 
of  the  tube  and  can  be  examined  microscopically. 

Gases  like  Hydrogen  to  Replace  the  Oxygen.— Several 
arrangements  for  passing  a  stream  of  hydrogen  through  the 
culture: 

Frankel  puts  in  the  test-tube  a  rubber  cork  containing  two 
glass  tubes,  one  reaching  to  the  bottom  and  connected  with  a 
hydrogen  apparatus,  the  other  very  short,  both  bent  at  right 
angles.  When  the  hydrogen  has  passed  through  ten  to  thirty 
minutes,  the  short  tube  is  annealed  and  then  the  one  in  connec- 
tion with  the  hydrogen  bottle,  and  the  gelatin  rolled  out  upon 
the  walls  of  the  tube  (Fig.  42). 


Fig.  42. — Frankel's  method  of 
making  anaerobic  cultures  (McFar- 
land). 


Fig.  43. — Buchner's  method  of 
making  anaerobic  cultures  (McFar- 
land). 


Use  of  Aerobic  Bacteria  to  Remove  the  Oxygen. — Roux 

inoculates  an  agar  tube  through  a  needle-thrust,  after  which 
semisolid  gelatin  is  poured  in  on  top.     When  the  gelatin  has 


74 


ESSENTIALS    OF    BACTERIOLOGY 


solidified,  the  surface  is  inoculated  with  a  small  quantity  of 
Bacillus  subtilis  or  some  other  aerobic  germ.  The  subtilis  does 
not  allow  the  oxygen  to  pass  by,  appropriating  it  to  itself. 

Buchner's  Method.— The  test-tube 
containing  the  culture  is  placed  within 
a  larger  tube,  the  lower  part  of  which 
contains  an  alkaline  solution  of  pyro- 
gallic  acid.  The  tube  is  then  closed 
with  a  rubber  stopper  (Fig.  43). 

Botkin's  Method.— Petri  dishes, 
uncovered,  are  placed  on  a  rack  under  a 
large  bell-jar,  into  which  hydrogen  gas 
is  conducted.  Alkaline  pyrogallic  acid 
is  placed  in  the  upper  and  lower  dishes 
to  absorb  what  oxygen  remains. 

Wright's  Method.— Applicable  to 
both  fluid  and  solid  media.  After  in- 
oculating the  test-tube,  the  plug,  which 
must  be  of  absorbent  cotton,  is  cut  off 
flush  with  the  extremity  of  the  tube, 
and  pushed  inward  for  a  distance  of  i 
cm.  It  is  then  impregnated  with  i  c.c. 
of  a  watery  solution  of  pyrogallic  acid 
and  T  c.c.  of  5  per  cent,  sodium  hydroxid 
solution.  A  tightly  fitting  rubber  stop- 
per is  inserted,  and  the  tube  is  then 
ready  for  incubation.  (See  Fig.  44.) 
Park's  Method.— An  Erlenmeyer 
flask  containing  the  medium  to  be  used 
is  boiled  in  a  water-bath  from  ten  to 
fifteen  minutes  to  drive  off  dissolved 
oxygen,  quickly  cooled,  and  inoculated. 

Hot  melted  paraffin  is  then  poured  into  the  flask,  which  forms 
a  layer  over  the  medium,  and  on  congealing,  provides  an  air- 
tight seal  which  does  not  adhere  to  the  glass  so  closely  as  to 
prevent  the  escape  of  any  gases  formed  by  the  bacterial 
growth. 


Fig.  44. — Wright's 
method  for  the  cultivation 
of  anaerobes. 


INFECTION  75 

CHAPTER  XIII 
INFECTION 

How  Bacteria  Cause  Disease. — Many  theories  have  been 
advanced  to  explain  the  action  of  bacteria  in  causing  disease, 
but  only  a  few  of  the  more  important  ones  can  be  taken  up  here. 
Nearly  all  the  changes  found  in  the  organs  of  the  body  are 
similar  to  those  produced  by  drugs  and  can  be  reproduced 
by  the  injection  of  bacterial  poisons. 

What  are  the  Conditions  Necessary  to  Produce  Infection? 

First,  as  to  the  infective  agent.  The  organism  must  have 
the  power  to  produce  disease.  It  must,  in  other  words,  be 
pathogenic.  A  non-pathogenic  bacterium  under  certain  con- 
ditions may  cause  disease,  but  this  is  not  an  infectious  disease; 
it  is  due  to  the  absorption  of  poisons  generated  outside  of  the 
body.  It  must  be  parasitic — have  the  power  of  growing  within 
the  body  of  an  animal. 

Essentially  an  infectious  disease  is  a  toxemia,  because  it 
depends  upon  poisons  or  toxins  produced  in  the  body.  Para- 
sitic or  infectious  bacteria  cause  disease  by  growing  in  the  animal 
organism  and  generating  products  therein  which  are  toxic. 
Saprophytic  bacteria  grow  outside  of  the  animal  organism  in 
dead  matter,  decaying  particles,  etc.,  and  they  may  give  rise 
to  products  which  also  are  toxic  to  the  animal  economy. 

Second,  the  toxins  or  poisons  elaborated  must  be  present  in 
sufficient  amount.  Undoubtedly  each  animal  organism  is  a  law 
unto  itself  in  regard  to  the  amount  of  poison  it  will  tolerate 
before  disease  is  actually  produced.  The  period  of  incubation 
can  be  explained  on  the  supposition  that  the  germ  requires  so 
much  time  to  elaborate  the  amount  of  toxin  necessary.  This 
time  period  varies  with  different  organisms,  some  carrying  the 
toxin  with  them  at  the  time  of  entry. 

Third,  the  animal  infected  must  be  susceptible.  Susceptibility 
varies  in  different  species  of  animals,  in  different  members  of 
the  same  species,  in  the  same  individual  at  different  times,  and 


76          ESSENTIALS  OF  BACTERIOLOGY 

in  the  same  individual  to  the  different  forms  of  disease  germs. 
Susceptibility  may  be  natural  to  the  race,  it  may  be  acquired,  it 
may  be  inherited.  Mice  are  naturally  susceptible  to  anthrax. 
Acquired  susceptibility  occurs  upon  exposure  to  conditions 
which  lower  vitality,  as  hunger,  cold,  advanced  age,  and  sur- 
gical shock.  Inherited  susceptibility  is  a  less  important  factor 
now  than  formerly.  Many  diseases  were  at  one  time  considered 
inherited  which  now  are  known  to  be  acquired  during  the  life- 
time of  an  individual.  Still,  certain  physical  characteristics, 
such  as  narrow  chest,  mouth-breathing,  etc. — clearly  inheritable 
characters — predispose  to  disease.  Given  a  susceptible  individ- 
ual and  an  infective  microorganism  producing  toxins  in  suffi- 
cient amount,  disease  is  certain  to  result. 

Fourth,  the  infecting  organism  must  gain  entrance  into  the 
tissues  or  find  lodgment  on  some  portion  of  the  body  that  has 
been  injured;  on  the  skin,  in  the  nose,  and  in  the  throat  are 
at  all  times  bacteria  that  are  more  or  less  pathogenic,  but 
they  fail  to  cause  disease  until,  through  a  traumatism  or 
lessened  resistance,  they  are  allowed  to  enter  the  tissues  or 
the  blood  and  then  they  act. 

Local  Effects  of  Bacteria. — By  mechanical  obstruction  from 
rapid  growth,  thrombosis,  with  its  consequences,  may  occur. 
Destruction  of  a  part  of  the  cells  of  a  tissue  with  necrosis  can 
arise  from  irritation,  as  from  a  foreign  body. 

General  Effects. — Sapremia,  when  toxic  products  of  local 
suppuration  are  absorbed  into  the  system.  Septicemia,  when 
the  infective  agent  itself  enters  the  blood-stream  and  causes 
general  disturbance. 

Suppurative  bacteria  are  those  which  give  rise  to  inflamma- 
tion and  suppuration  locally  at  the  point  of  entrance,  and 
secondarily  through  metastasis.  Any  organism  may  cause 
suppuration,  but  a  number  are  peculiarly  inclined  to  give  rise 
to  pus,  and  are  known  as  pyogenic  organisms. 

Specific  Bacteria. — Infective  bacteria  are,  as  a  rule,  specific, 
the  particular  toxin  having  a  specific  action  and  causing  a  dis- 
ease peculiar  to  the  microorganism.  Thus  typhoid  fever  is  a 
disease  distinctly  different  from  tuberculosis;  the  infective 


INFECTION  77 

organisms  are  distinct  and  the  poisons  they  produce  have 
specific  characteristics. 

The  Nature  of  Toxins. — Very  similar  to  the  venom  of  ser- 
pents; highly  poisonous  in  minute  doses  (ToVo  g^111  of  tetanus 
toxin  will  kill  a  horse  weighing  600  kilos — 1200  pounds).  At 
first  toxins  were  called  ptomains,  or  cadaveric  alkaloids;  but 
this  term  is  applied  now  to  such  poisons  as  have  a  basic  nature 
and  arise  in  decomposing  meat,  cheese,  and  cream  as  a  result 
of  chemical  change  in  the  material,  the  bacteria  causing  the 
change.  Then  they  were  called  toxalbumins,  and  were  supposed 
to  belong  to  an  albumin  series;  but  when  the  bacteria  are  grown 
in  non-albuminous  media,  the  toxins  correspond  more  in  their 
chemical  composition  to  &  ferment,  and  therefore  it  is  supposed 
that  the  albumin  part  of  the  toxin  is  furnished  by  the  blood  or 
albuminous  media  in  which  it  is  formed.  The  term  toxin  is  to 
be  preferred  in  speaking  of  bacterial  poisons. 

Toxins  may  be  of  two  sorts:  (a)  Chiefly  within  the  bodies  of 
the  bacteria,  so  that  they  are  set  free  by  the  disintegration  of 
the  organisms.  This  group  comprises  most  of  the  pathogenic 
bacteria  and  must  be  combated  by  the  use  of  antibacterial 
serums,  (b)  The  poisons  seem  to  be  excreted  by  the  bacteria 
and  are  found  in  the  surrounding  medium.  Antitoxic  serums 
are  applicable  to  this  group,  which  includes  the  bacilli  of  diph- 
theria and  tetanus.  Welch  has  suggested  that  even  bacteria 
which  do  not  appear  to  form  toxins  in  artificial  cultures  may 
do  so  in  the  human  body.  In  the  effort  to  adapt  themselves 
to  their  environment  and  resist  the  hostile  agencies  of  the  body 
they  produce  the  poisons  we  call  toxins.  (For  method  of  pro- 
duction of  an  antitoxin,  see  article  on  Diphtheria.) 

Toxins  are  not  stable;  they  are  uncrystallizable,  soluble  in 
water;  they  are  allied  to  albumose  in  that  they  are  precipitated 
by  alcohol  and  ammonium  sulphate. 

Aggressin. — A  name  given  to  a  form  of  toxin  developed  in  the 
animal  body  that  has  the  power  of  rendering  a  germ  more  active 
(aggressive) .  If  such  a  toxin  is  obtained  from  an  experiment 
animal  and  added  to  a  growth  of  bacteria,  it  makes  that  culture 
particularly  virulent.  Aggressins  are  supposed  to  have  a  par- 
alyzing influence  on  the  phagocytes, 


78  ESSENTIALS    OF    BACTERIOLOGY 

CHAPTER  XIV 

IMMUNITY 

Immunity,  as  distinguished  from  susceptibility,  is  merely  a 
relative  term,  as  no  animal  is  absolutely  immune  under  all  condi- 
tions. It  is  merely  less  susceptible,  and  some  animals  are  by 
nature  or  can  by  artificial  means  be  rendered  so  slightly  sus- 
ceptible that  to  all  practical  purposes  they  are  immune — that  is, 
capable  of  resisting  an  attack  of  the  particular  disease  against 
which  they  are  said  to  be  immune. 

Natural  Immunity.— The  goat  and  dog  are  considered 
naturally  immune  to  tuberculosis.  Algerian  sheep  are  resistant 
to  anthrax;  other  varieties  are  susceptible. 

The  field-mouse  is  susceptible  to  glanders;  the  white  mouse 
is  ordinarily  immune.  House  mice  are  susceptible  to  mouse 
septicemia;  field-mice  are  immune. 

Acquired  Immunity. — Immunity  can  be  acquired  in  many 
ways.  Active  and  passive  immunity  are  varieties. 

Active  immunity  can  be  acquired  from  an  attack  of  the  disease; 
such  infectious  diseases  as  measles,  scarlatina,  and  whooping- 
cough  usually  confer  immunity  from  future  attacks.  Some 
diseases  render  the  individual  immune  for  only  a  short  period. 

Immunity  from  Inoculation  with  Attenuated  or  Weak- 
ened Cultures  of  Bacteria. — Vaccination  is  an  example. 
Haffkine's  cholera  vaccines  and  Pasteur's  vaccines  of  anthrax 
and  chicken  cholera  are  likewise  examples  of  this  method. 

Attenuation  is  produced  as  follows:  Successive  cultivation  in 
artificial  media  destroys  the  virulence  of  bacteria.  Old  cultures 
are  less  virulent  than  fresh  ones.  Virulence  is  lessened  by 
passing  the  cultures  through  animals  that  are  less  susceptible 
or  entirely  immune.  The  cautious  use  of  chemicals  and  sun- 
light lessens  virulence.  Heat  is  an  effective  agent.  An  anthrax 
culture  exposed  to  a  temperature  of  42.6°  C.  for  twenty  days 
will  prove  destructive  only  to  animals  no  larger  than  mice. 
Prolonged  exposure  to  oxygen  weakens  the  germs. 


IMMUNITY  79 

Immunity  Through  Inoculations  of  Small  Doses  of  Very 
Virulent  Microorganisms. — A  graduated  resistance  to  the 
disease  is  reached  somewhat  after  nature's  method.  By  succes- 
sive inoculations  with  increased  doses  of  the  virus  an  immunity 
is  often  reached  sufficient  to  withstand  ten  times  the  lethal 
dose.  A  poison-habit  is  thus  acquired. 

Increased  virulence  is  produced  as  follows:  The  cultures 
may  be  greatly  increased  in  virulence  by  successive  cultivation 
through  animals,  and  gradually  changing  from  smaller  animals 
to  larger,  until  an  amount  of  the  culture  that,  at  the  outset, 
would  not  destroy  a  guinea-pig,  becomes  finally  virulent  for 
chickens  and  dogs. 

Immunity  Through  Injections  of  the  Sterilized  Products 
of  Bacteria. — Cultures  sterilized  by  heat  or  filtration  through 
germ-filters  still  contain  the  chemical  products  of  bacteria — 
the  toxins;  and  when  these  are  injected  in  gradually  increased 
doses,  the  same  immunity  is  obtained  as  with  the  bacteria 
themselves. 

Passive  Immunity. — The  blood-serum  and  tissues  generally 
of  animals  rendered  immune  in  the  ways  described  above,  when 
injected  into  susceptible  animals,  render  them  immune  against 
the  same  infection.  This  has  been  called  passive  immunity,  but 
there  is  no  strong  reason  why  this  term  should  be  used.  The 
blood-serum  of  immune  animals  is  simply  another  means  for 
immunization.  It  is  less  permanent  than  the  other  forms  of 
immunization,  but  it  appears  very  soon  after  the  injection,  and 
in  a  modified  form  has  a  curative  action  even  when  the  symp- 
toms of  the  infection  are  already  present  in  the  system. 

Inherited  Immunity. — An  immunity  to  disease  acquired  dur- 
ing the  lifetime  of  the  parents  is  probably  never  transmitted 
to  the  offspring,  though  the  mother  may  transmit  a  temporary 
immunity  to  the  child  in  utero,  or  the  child  itself  may  have 
been  subjected  to  the  infection  at  the  same  time  with  its  mother. 
But  this  cannot  be  called  inherited. 

Theories  of  Immunity. — Several  older  theories  only  need 
to  be  mentioned,  as  they  are  no  longer  tenable.  They  are 
the  exhaustion  theory  of  Pasteur,  the  retention  theory,  and  the 


8o  ESSENTIALS    OF    BACTERIOLOGY 

humeral  theory.  "  At  present,  modifications  of  Metchnikoff's 
phagocytic  theory  and  Ehrlich's  side-chain  theory  seem  the 
most  plausible. 

Phagocytic  or  Cellular  Theory. — Metchnikoff  elaborated  this 
after  his  study  on  inflammation.  Phagocytosis  occurs  in 
animals  when  subjected  to  the  action  of  an  irritant.  The  leu- 
kocytes are  attracted  to  the  injured  spot  and  envelop  the  irri- 
tating substance,  be  it  bacteria  or  dead  matter.  The  theory 
given  out  at  first  was  that  if  the  leukocytes  conquer  the  bacteria, 
immunity  results;  if  the  bacteria  eat  up  the  leukocytes,  disease 
occurs.  Modified  to  suit  other  conditions,  as,  for  instance, 
the  germicidal  properties  of  serum  freed  from  its  cellular  ele- 
ments, Metchnikoff  states  that  at  times  phagolysis — that  is, 
breaking  up  or  solution  of  the  phagocytes — takes  place,  and 
the  fluids  in  which  these  cells  are  dissolved  become  charged 
with  the  powers  originally  present  in  the  phagocytes.  Chemo- 
taxis  is  the  term  applied  to  the  attraction  of  bacteria  for  the 
leukocytes,  and  is  supposed  to  be  chemical  in  its  nature.  The 
phagocytic  cells  comprise:  (a)  The  polymorphonuclear  leu- 
kocytes of  the  blood,  termed  microphages,  and  (6)  a  group 
called  macrophages,  which  includes  all  other  cells  having 
phagocytic  properties,  such  as  leukocytes  other  than  the  poly- 
morphonuclears,  endothelial  cells,  and  connective-tissue  cor- 
puscles. When  these  cells  are  injured,  they  set  free  their  diges- 
tive ferments,  known  as  microcytases  and  macrocytases  respect- 
ively, which  correspond  to  the  alexins  of  Ehrlich. 

Ehrlich's  Side-chain  Theory. — This  derives  its  name  from  the 
fact  that  it  presents  an  analogy  to  what  happens  in  the  benzol 
ring  of  organic  chemistry  when  its  replaceable  atoms  of  hydro- 
gen are  substituted  by  "side  chains"  of  more  or  less  complex 
nature.  The  molecule  of  protoplasm  is  supposed  to  consist  of 
a  central  atom  group  provided  with  a  large  number  of  side 
chains  which  subserve  the  vital  processes  of  the  molecule  by 
combining  with  other  organic  molecules.  These  side  chains 
are  called  receptors,  and  are  of  many  different  kinds,  so  as  to  fit 
them  for  combination  with  many  different  varieties  of  extrane- 
ous groups.  Bacterial  toxins  contain  two  groups:  (i)  The 


IMMUNITY  8 1 

haptophores,  by  which  the  toxin  molecule  can  become  joined 
to  the  cell;  (2)  the  toxophores,  by  virtue  of  which  it  can  attack 
the  protoplasm  after  having  been  fixed  to  it  by  the  hapto- 
phore.  If  the  attack  on  the  molecule  is  not  too  severe,  this  is 
stimulated  into  overactivity  and  throws  out  an  abnormal  number 
of  receptors,  some  of  which  (the  haptins)  become  detached 
and  are  capable  of  uniting  with  free  haptophores  and  prevent- 
ing their  combination  with  the  protoplasm  of  the  molecule. 
In  other  words,  they  represent  the  antitoxin. 

Hemolytic  Serum. — The  blood-serurn  of  some  animals 
has  the  power  of  destroying  or  dissolving  the  red  corpuscles 
of  another  animal  of  different  species.  This  power  can  be 
increased  by  repeated  injections  of  blood-corpuscles,  and  is 
allied  to  the  bactericidal  power. 

Bacteriolysis  is  the  destruction  of  the  bacterial  cells  by  the 
blood-serum,  and  is  probably  effected  in  a  somewhat  different 
manner.  Antibacterial  serums  are  effective  through  the  com- 
bined activities  of  a  destructive  element,  the  "complement" 
(alexin  or  cytase),  and  an  "  immune  body"  (amboceptor) ,  which 
serves  the  function  of  joining  the  complement  to  the  bacterial 
molecule.  These  two  bodies  differ  markedly  in  their  properties 
—for  example,  the  complement  is  destroyed  at  60°  C.,  while  the 
immune  body  is  very  resistant. 

It  is  not  stated  what  cells  are  the  sources  of  these  various 
antibodies,  but  probably  any  cell  capable  of  being  attacked  by 
a  toxin  is  also  capable  of  responding  by  the  production  of  anti- 
substances. 

Lysins. — The  substances  producing  destruction  of  bacteria 
are  called  lysins.  Normal  blood-serum  is  bacteriolytic  to  a 
slight  degree,  but  during  infection  produces  lysins  specific  for 
the  germ  in  question. 

Agglutinins. — These  are  bodies  formed  in  the  blood-serum 
in  response  to  the  stimulation  of  certain  bacteria,  such  as  the 
typhoid  bacillus,  Bacillus  coli  communis,  Micrococcus  meli- 
tensis,  the  bacillus  of  dysentery,  the  cholera  spirillum,  etc. 
When  such  a  serum  is  added  to  cultures  of  the  particular  organ- 
ism concerned,  the  bacteria  become  clumped  in  motionless 


82  ESSENTIALS    OF    BACTERIOLOGY 

masses.  A  modified  form  of  agglutination,  in  which  long 
strings  of  bacteria  are  formed,  is  known  as  the  "thread"  reac- 
tion. 

Agglutinins  are  of  different  kinds,  and  they  are  not  an  indi- 
cation of  immunity.  A  serum  may  give  a  strong  agglutinating 
reaction,  but  have  little  or  no  bactericidal  power.  The  power 
varies  from  day  to  day — it  is  strongest  in  the  blood-serum,  but 
is  found  in  other  fluids  of  the  body. 

Agglutinogen  is  the  name  given  to  the  substance  on  the 
bacteria,  while  agglutinin  is  the  term  restricted  to  the  sub- 
stance in  the  serum. 

Precipitins. — Animals  immunized  to  certain  bacteria  or  to 
albumins  of  different  sorts  form  bodies  which  cause  the  blood- 
serum  to  give  a  precipitate  when  added  to  cultures  of  these 
organisms  or  fluids  containing  the  specific  albumin.  The 
phenomenon  has  found  forensic  application  in  the  identifica- 
tion of  blood-stains. 

Opsonins. — Much  work  has  been  done  in  the  last  few  years 
to  harmonize  the  various  theories  on  immunity,  and  a  new  one, 
advanced  by  Wright  and  Douglas  in  1905,  seems  to  have  much 
in  its  favor. 

Briefly  stated,  the  leukocytes  have  no  power  in  themselves 
to  act  upon  bacteria,  but  derive  this  property  from  the  blood- 
serum.  In  normal  blood-serum  Wright  maintains  there  is 
a  body  which  he  calls  "opsonin,"  which  becomes  fixed  to  the 
bacterial  cells  and  makes  them  subject  to  phagocytosis.  This 
power  of  the  serum  is  greater  the  more  immune  the  animal 
furnishing  it  is. 

It  can  be  increased  by  whatever  increases  immunity;  in 
other  words,  it  is  coincident  with  immunity. 

History. — Denys  and  Leclij  (1895)  proved  that  the  serum  of 
a  vaccinated  rabbit  altered  bacteria  so  as  to  permit  their  inges- 
tion  by  leukocytes. 

Mennes  (1897)  showed  that  this  modification  resides  in  the 
serum  and  not  in  the  leukocytes. 

Leishman  (1902)  devised  a  method  of  estimating  the  phago- 
cytic  power  of  serums. 


IMMUNITY  83 

Wright  (1902)  showed  that  sterile  cultures  of  certain  micro- 
cocci  given  in  small  doses  raised  the  agglutinating  power  of 
the  blood  to  that  particular  organism.  Wright  (1903)  using 
Leishman's  method,  found  that  the  phagocytic  index  is  raised 
by  the  injection  of  bacterial  products. 

Wright  and  Douglas  (1903)  gave  the  name  of  opsonin  (from 
opsono,  I  prepare  the  food  for)  to  this  element  in  the  blood, 
arguing  that  in  some  way  the  bacteria  are  acted  upon  by  this 
substance,  making  them  more  readily  digested. 

Opsonic  Index. — The  degree  of  immunity  of  a  given  blood- 
serum,  as  compared  with  that  taken  from  a  healthy  individual, 
considered  as  a  unit,  is  called  the  index. 

The  activity  of  the  leukocytes  toward  an  emulsion  of  bacteria 
is  noted  first  when  washed  with  normal  serum,  and  then  when 
washed  with  serum  from  an  affected  person.  If,  in  the  one 
instance,  in  50  cells  an  average  of  3  is  obtained,  and,  in  the 
other  instance,  the  average  is  only  ij,  the  index  is  then  said 
to  be  0.5,  or  one-half  of  the  normal.  This  index  is  measured 
from  day  to  day  with  the  course  of  the  disease  and  treatment, 
and  charted  just  as  the  temperature  is. 

A  negative  phase,  that  is,  a  decreased  opsonic  index,  often 
follows  the  use  of  bacterial  products;  then  comes  a  positive 
phase,  or  a  rise  in  phagocytosis.  Injections  of  vaccines  are 
harmful  if  repeated  before  the  negative  phase  has  worn  off. 

Summary  of  Immunity  Theories. — At  this  writing  the 
following  ideas  concerning  immunity  seem  to  be  accepted: 

The  blood-serum  contains  substances — opsonins — which 
envelop  or  attach  themselves  to  bacterial  cells,  rendering  them 
fit  for  leukocytic  digestion — phagocytosis.  The  growth  of 
bacteria  in  the  body  stimulates  the  production  of  antibodies, — 
antitoxins, — and  the  activity  of  the  blood-cells  likewise  calls 
forth  an  increased  activity  of  the  germ  and  an  increase  in  toxin. 
The  theory  of  Ehrlich,  or  chemical  explanation  of  the  action 
of  antitoxin,  is  still  a  subject  of  controversy. 


84          ESSENTIALS  OF  BACTERIOLOGY 

CHAPTER  XV 
EXPERIMENTS  UPON  ANIMALS 

THE  smaller  rodents  and  birds  are  the  ones  usually  employed 
for  inoculation,  as  rabbits,  guinea-pigs,  rats,  mice,  pigeons, 
and  chickens,  sometimes  monkeys.  These  are  preferred, 
because  easily  acted  upon  by  the  various  bacteria,  readily 
obtained,  and  not  expensive. 

The  white  mouse  is  very  prolific  and  easily  kept,  and  is  there- 
fore a  favorite  animal  for  experiment.  It  lives  well  upon  a  little 
moistened  bread.  A  small  box,  perforated  with  holes,  is  filled 
partly  with  sawdust,  and  in  this  ten  to  twelve  mice  can  be  kept. 
When  the  female  becomes  pregnant,  she  should  be  removed  to 
a  glass  jar  until  the  young  have  opened  their  eyes,  because  the 
males,  which  have  not  been  raised  together,  are  apt  to  attack 
each  other. 

Guinea-pigs. — When  guinea-pigs  have  plenty  of  light  and 
air,  they  multiply  rapidly.  Therefore  it  is  best  to  have  them  in 
some  large  stall  or  inclosure.  They  can  be  fed  upon  all  sorts  of 
vegetables  and  grasses,  and  require  but  little  attention. 

Methods  of  Inoculation. — /.  Inhalation. — Imitating  the 
natural  infection,  either  by  loading  an  atmosphere  with  the 
germs  in  question  or  by  administering  them  with  a  spray. 

//.  Through  skin  or  mucous  membrane. 

III.  With  the  food. 

Method  of  Cutaneous  Inoculation. — The  ear  of  mice  is 
best  suited  for  this  procedure.  A  small  abrasion  made  with 
the  point  of  a  lancet  or  needle,  which  has  been  dipped  in  the 
virus.  The  animal  is  then  separated  from  the  rest  and  placed 
in  a  glass  jar,  which  is  partly  filled  with  sawdust  and  covered 
with  a  piece  of  wire  gauze. 

Subcutaneous. — The  root  of  the  tail  of  mice  is  used  for  this 
purpose.  The  hair  around  the  root  of  the  tail  is  clipped  off, 
and  with  a  pair  of  scissors  a  very  small  pocket  is  made  in  the 
subcutaneous  connective  tissue,  not  wounding  the  animal  any 


EXPERIMENTS  UPON  ANIMALS  85 

more  than  absolutely  necessary,  avoiding  much  blood.  The 
material  is  placed  upon  a  platinum  needle  and  introduced  into 
the  pocket;  solid  bodies,  with  a  forceps. 

To  hold  the  mouse  still  while  the  operation  is  going  on  a 
little  cone  made  of  metal  is  used.  The  mouse  just  fits  in 
here.  There  is  a  slit  along  the  top  in  which  the  tail  can  be 
fastened,  and  thus  the  animal  is  secure  and  immobile. 

Intravenous  Injections.— Rabbits  are  very  easily  injected 
through  the  veins.  Mice  are  too  small. 


Fig.  45. — Method  of  making  an  intravenous  injection  into  a  rabbit. 
Observe  that  the  needle  enters  the  posterior  vein  from  the  hairy  surface 
(McFarland). 

The  ear  of  the  rabbit  is  usually  taken.  It  is  first  washed 
with  T  :  2000  bichlorid,  which  not  only  disinfects,  but  .also 
makes  the  vessels  appear  more  distinct.  The  base  of  the  ear  is 
compressed  to  swell  the  veins.  Then  a  syringe,  like  the  one 
used  for  the  injection  of  " tuberculin,"  a  Koch  syringe,  which 
can  be  easily  sterilized,  is  filled  with  the  desired  amount  of 
virus  and  slowly  injected  into  any  one  of  the  more  prominent 
veins  present  (Fig.  45). 


86  ESSENTIALS    OF    BACTERIOLOGY 

Intraperitoneal  Injection. — This  is  used  with  guinea-pigs 
mostly.  The  abdominal  wall  is  pinched  up  through  its  entire 
thickness,  and  the  needle  of  the  syringe  thrust  directly  through, 
so  that  it  appears  on  the  other  side,  then  the  fold  let  go,  the 
needle  withdrawn  just  far  enough  so  as  to  be  within  the  cavity. 

Inoculation  in  the  Eye. — The  anterior  chamber  and  the 
cornea  are  the  two  places  used.  The  rabbit  is  fixed  upon  a 
board,  the  eyelids  held  apart  and  head  held  still  by  an  assistant. 
A  small  cut  is  made  in  the  cornea,  a  few  drops  of  cocain  having 
first  been  introduced  in  the  eye.  The  material  is  passed 
through  the  opening  with  a  small  forceps,  and  with  a  few  strokes 
of  a  spoon  it  is  pushed  in  the  anterior  chamber. 

For  the  cornea  a  few  scratches  made  in  the  corneal  tissue 
will  suffice;  the  material  is  then  gently  rubbed  in. 

Inoculation  of  the  Cerebral  Membranes. — The  skin  and 
aponeurosis  cut  through  where  the  skull  is  the  thinnest.  Then 
the  bone  carefully  trephined,  and  the  dura  exposed.  In  rabies 
inoculation,  the  syringe  containing  the  hydrophobic  virus  pierces 
the  dura  and  arachnoid,  and  the  virus  is  discharged  beneath  the 
latter. 

Intratracheal. — The  bacteria  can  be  introduced  directly 
into  the  trachea,  thus  coming  in  contact  with  the  lungs. 

Intraduodenal. — Cholera  germs  are  injected  into  the  intes- 
tines after  they  have  been  exposed,  by  carefully  opening  the 
abdomen.  This  is  done  in  order  to  avoid  the  action  of  the 
gastric  juice. 

Celloidin  sacs  of  small  size  are  sometimes  used  to  introduce 
living  cultures  of  bacteria  into  the  bodies  of  animals  without 
their  coming  into  direct  contact  with  the  tissues. 

Obtaining  Material  from  Infected  Animals.— The  animal 
should  be  skinned,  or  the  hairs  plucked  out,  before  it  is  washed 
— at  least  the  portion  where  the  incision  is  to  be  made.  Then 
the  entire  body  is  washed  in  sublimate.  Two  sets  of  instru- 
ments are  required — one  for  coarser  and  one  for  finer  work: 
the  one  sterilized  in  the  flame;  the  other,  to  prevent  being 
damaged,  heated  in  a  hot-air  oven. 

The  animal,  the  mouse,  for  example,  is  stretched  upon  a 


OPSONIC   TECHNIC  87 

board,  a  nail  or  pin  through  each  leg,  and  the  head  fixed  with 
a  pin  through  the  nose.  The  skin  is  dissected  away  from  the 
belly  without  exposing  the  intestines.  Then  the  ribs  being 
laid  bare,  the  sternum  is  lifted  up,  and  the  pericardium  exposed. 
A  platinum  needle  dipped  into  the  heart  after  the  pericardium 
has  been  slit  will  give  sufficient  material  for  starting  a  culture. 
If  the  other  organs  are  to  be  examined,  further  dissection  is 
made.  If  the  intestines  were  first  to  be  looked  at,  they  would 
be  laid  bare  first. 

In  this  manner  material  is  obtained  and  the  results  of  inocu- 
lation noted. 

Frequent  sterilization  of  the  instruments  is  desirable. 

Koch's  Rules  in  Regard  to  Bacterial  Cause  of  Disease.— 
Before  a  microbe  can  be  said  to  be  the  cause  of  a  disease,  it 
must — 

First,  be  found  in  the  tissue  or  secretions  of  the  animal  suf- 
fering from,  or  dead  with,  the  disease. 

Second,  it  must  be  cultivated  outside  of  the  body  on  artificial 
media. 

Third,  a  culture  so  obtained  must  produce  the  disease  in 
question  when  it  is  introduced  into  the  body  of  a  healthy  animal. 

Fourth,  the  same  germ  must  then  again  be  found  in  the  animal 
so  inoculated. 


CHAPTER  XVI 
OPSONIC  TECHNIC 

Method  of  Counting  Bacteria  in  a  Culture.— In  the  use 

of  protective  vaccines,  a  method  of  counting  the  bacteria  in  a 
given  amount  of  serum  or  emulsion  has  come  into  practice, 
especially  in  the  opsonic  index  treatment. 

First,  the  number  of  red  cells  in  a  cubic  millimeter  of  blood 
is  measured;  then,  in  a  capillary  tube  (Wright  pipet),  blood 


88  ESSENTIALS    OF    BACTERIOLOGY 

is  sucked  up  to  a  certain  mark,  and,  with  an  air-bubble  between, 
a  diluted  suspension  of  bacterial  culture  is  measured  into  the 
same  tube.  From  this  mixture,  one  drop  is  blown  out  on  to  a 
slide  and  stained  according  to  Leishman's  method,  and  the 
bacteria  compared  to  the  blood-cells.  If  the  observer's  blood 
contained  5,000,000  red  cells  in  a  cubic  millimeter,  and  with  a 
i  :  3  dilution  of  the  bacteria,  the  bacteria  are  equal  in  number 
to  the  red  cells,  in  the  undiluted  culture  each  cubic  millimeter 
would  contain  15,000,000  bacterial  cells. 

Method  of  Measuring  Phagocytosis — the  Opsonic  Tech- 
nic. — First,  Preparation  of  Bacterial  Emulsion. — The  culture 


Fig.  46. — Diagram   of   phagocytosis.     Staphylococci   in   polynuclear   leuko- 
cytes. 

from  an  agar  slant  is  rubbed  in  a  watch-crystal  with  0.85  per 
cent,  saline  solution,  and  the  mixture  centrifugalized  to  sedi- 
ment any  masses  undissolved.  The  emulsion  should  then  be 
just  very  slightly  cloudy,  and  will  contain  about  10,000,000,000 
bacteria  per  cubic  centimeter. 

Second,  Preparation  of  Leukocytes. — Healthy  blood-cells  are 
used — preferably  the  observer's.  At  least  ten  drops  of  blood 
are  mixed  in  a  test-tube  containing  3  c.c.  warm  normal  salt 
solution,  to  which  1.5  per  cent,  sodium  citrate  has  been  added; 
this  is  then  centrifuged,  the  supernatant  fluid  removed  down 
to  the  layer  of  leukocytes,  and  an  equal  quantity  of  normal  salt 


OPSONIC  TECHNIC  89 

solution  added  and  again  the  mixture  centrifuged,  the  fluid 
removed,  leaving  the  leukocytes  and  red  cells,  which  is  known 
as  the  leukocyte  emulsion  or  "washed  leukocytes" 

Third,  Preparation  of  the  Serums. — Small  capsules  made  from 
glass  tubing  (see  Fig.  47)  are  used  to  obtain  the  blood  from  a 
normal  or  healthy  person;  the  blood  by  attraction  is  made 
to  fill  a  capsule,  the  end  is  sealed  with  wax,  and  the  corpuscles 
separated  by  the  centrifuge.  A  capsule  with  blood  from  an 
infected  person  is  obtained,  and  the  serum  separated  in  the 
same  way. 

Fourth,  Mixture  of  the  Emulsion,  the  Leukocytes,  and  the 
Serum. — This  is  performed  by  drawing  into  a  pipet  similar 
portions  of  the  emulsion,  the  leukocytes,  and  the  serum,  mixing 


Fig.  47. — Wright's  blood-capsule.     Illustration  shows  technic  of  filling  same. 

a  drop  of  each  on  a  slide.  The  mixture  is  then  drawn  up  again 
into  the  tube,  the  end  of  which  is  now  sealed  and  the  tube  placed 
in  an  incubator  at  37°  C.  for  fifteen  minutes.  The  end  is 
broken,  and  a  drop  of  the  mixture  is  distributed  evenly  on  a 
glass  slide  and  stained.  The  same  procedure  is  carried  on 
with  the  serum  of  the  infected  person,  but  using  the  normal 
leukocytes. 

Under  the  microscope  the  number  of  bacteria  in  the  proto- 
plasm of  at  least  fifty  polynuclear  cells  are  counted  (see 
Fig.  46)  in  the  mixture  with  the  normal  serum  and  in  the 
mixture  with  the  infected  serum,  and  an  average  for  each 
obtained;  the  proportion  that  the  diseased  bears  to  the  normal 
is  the  opsonic  index. 


9° 


ESSENTIALS    OF    BACTERIOLOGY 


In  this  work  the  following  special  apparatus  are  required : 

1.  A  Wright's  tube  for  collecting  the  blood. 

2.  Four  c.c.  test-tubes  7  cm.  long  and  9  mm.  in  diameter, 
to  hold  the  washed  leukocytes. 


Fig.  48. — Tubes  utilized  in  testing  agglutinating  and  sedimenting  proper- 
ties of  serum:  a,  Leukocyte  meter  pipet;  b,  V-shaped  tube;  c,  capillary  pipet; 
d,  Wright's  sedimentation  tube. 

3.  Suction  pipets,  either  Gowers'  graduated  pipet,  as  used 
in  blood  examinations,  or  a  special  device  of  Wright's  (see 
d,  Fig.  48). 


PART   II 
SPECIAL  BACTERIOLOGY 


CHAPTER  XVII 
NON-PATHOGENIC  BACTERIA 

Special  Bacteriology. — Under  this  head  the  chief  character- 
istics of  individual  bacteria  will  be  detailed,  pathogenic  and  non- 
pathogenic  being  the  main  divisions.  The  division  is  not  a 
strict  one,  under  certain  conditions  bacteria  ordinarily  non- 
pathogenic  may  become  pathogenic. 

Non-pathogenic  Bacteria. — The  list  of  non-pathogenic 
bacteria  is  a  long  one,  and  is  being  added  to  continually. 

Bacillus  Prodigiosus  (Ehrenberg) .— This  bacillus,  for- 
merly called  micrococcus,  is  very  common,  and  one  of  the 
first  noticed,  because  of  the  brilliant  red  pigment  it  forms 
on  vegetables  and  starchy  substances.  "The  bleeding  host" 
miracles  are  said  to  have  been  due  to  it. 

Form. — Short  rods,  often  in  filaments,  without  spores. 

Immobile. — Has  no  automatic  movements. 

Facultative  anaerobic,  that  is,  it  can  grow  without  air;  but 
the  pigment  requires  oxygen  to  show  itself. 

Growth. — Gelatin.     Liquefy  rapidly. 

Colonies. — At  first  white,  round  points  with  smooth  edge, 
appearing  brown  under  microscope,  but  soon  changing  to  red. 

Stab-cultures. — Red  pigment  develops  on  the  surface,  the 
growth  occurring  all  along  the  line. 

Potato  is  well  suited  to  the  growth,  the  pigment  developing 
after  twelve  hours.  A  gar  and  blood-serum  well  suited  for  growth. 

Temperature. — Grows  best  at  25°  C. 

91 


Q2  ESSENTIALS    OF    BACTERIOLOGY 

Varieties. — By  exposure  to  heat  of  brood-oven  during  several 
generations  the  power  to  produce  pigment  can  be  temporarily 
abolished. 

The  Pigment. — A  pigment- forming  body  is  created  by  the 
bacillus,  and  the  action  of  oxygen  upon  it  produces  the  color. 
It  is  insoluble  in  water,  slightly  soluble  in  alcohol  and  ether; 
acids  fade  it,  alkalis  restore  the  color.     The  pigment  resembles 
fuchsin,  presenting  the  same  metallic  luster. 
Gases. — A  trimethylamin  odor  arises  from  all  cultures. 
Stain. — With  all  dyes  in  the  ordinary  way. 
Bacillus  Indicus   (Koch). — Synonym. — Micrococcus  Indi- 
cus. 

Origin. — Found  in  the  stomach  of  an  Indian  ape. 
Form. — Short  rods  with  rounded  ends.     No  spores.    Auto- 
matic movements  present;  facultative  anaeroHn. 
Growth. — Gelatin. — Liquefy  rapidly. 

Colonies. — Round  or  oval  granular  margins;  brilliant  red 
pigment. 

Stab-cultures. — The  pigment  shows  itself  on  the  surface. 
Grows  well  on  all  media. 

Temperature. — Grows  best  at  35°  C. 

Action  on  Animals. — In  very 
large  quantities,  if  injected  into 
the  blood,  a  severe  and  fatal  gas- 
tro-enteritis  can  be  produced. 
Stain. — Takes  all  dyes. 
Bacillus  Mesentericus  Vul- 
gatus. — The     common    potato 
bacillus  of  Flugge  (Fig.  49) . 

Habitat. — Surface  of  the  soil, 
on  potatoes,  and  in  milk. 
Fig.  49.-Coiony  of  Bacillus  mes-        Form.— Small  thick  rods  with 

entencus  vulgatus. 

rounded  ends,  often  in    pairs. 

Properties. — Very  motile;  produces  abundant  spores;  liquefy 
gelatin;  diastatic  action. 
Growth. — Rapid. 
Plate  Colonies. — Round,  with  transparent  center  at  first,  then 


NON-PATHOGENIC    BACTERIA 


93 


becoming  opaque.  The  border  is  ciliated;  little  projections 
evenly  arranged. 

Potato. — A  white  covering  at  first,  which  then  changes  to  a 
rough  brown  skin;  the  skin  can  be  detached  in  long  threads. 

Temperature. — Spores  at  ordinary  temperature. 

Spores.— Are  very  resistant;  are  colored  in  the  manner 
described  in  first  part  of  the  book  for  spores  in  general. 

Bacillus  Megaterium  (de  Bary)  (Fig.  50).— Origin.— 
Found  on  cooked  cabbage  and  garden-soil. 


Fig.  50. — Bacillus  megaterium,  with  spores. 

Eorm. — Large  rods,  four  times  as  long  as  they  are  broad, 
2.5  /•*.  Thick  rounded  ends.  Chains  with  ten  or  more  members 
often  formed;  granular  cell  contents. 

Properties. — Abundant  spore  formation;  very  slow  movement; 
slowly  dissolves  gelatin. 

Growth. — Strongly  aerobic;  grows  quickly  and  best  at  a 
temperature  of  20°  C. 

Plate  Colonies. — Small,  round,  yellow  points  in  the  depth  of 
the  gelatin.  Under  microscope,  irregular  masses. 

Stab-culture. — Funnel-shaped  from  above  downward. 

Potato. — Thick  growth  with  abundance  of  spores. 

Bacillus  Ramosus. — Synonyms. — Bacillus  mycoides 
(Fliigge);  Wurzel  or  root  bacillus. 


94  ESSENTIALS    OF    BACTERIOLOGY 

Origin. — In  the  upper  layers  of  garden  or  farm  grounds  and 
in  water. 

Form. — Short  rods,  with  rounded  ends,  about  three  times 
as  long  as  they  are  thick;  often  in  long  threads  and  chains. 

Properties. — Large,  shining,  oval  spores;  a  slight  movement; 
liquefy  gelatin. 

Growth. — At  ordinary  temperature,  with  plentiful  supply  of 
air. 

Plate  Colonies. — Look  like  roots  of  an  old  tree  gnarled 
together,  radiating  from  a  common  center.  Liquid  on  surface. 

Stab-culture. — Soon  a  growth  occurs  along  the  needle-track, 
and  the  whole  resembles  a  pine  tree  turned  upside  down.  The 
gelatin  then  becomes  liquid,  a  thin  skin  floating  on  top,  and 
small  flakes  lying  at  the  bottom. 

Stroke-culture. — Feathery  resemblance  is  produced. 

Staining. — Spores  stain  readily  with  the  ordinary  spore  stain. 

Bacterium  Zopfii  (Kurth)  .—Origin.— Intestines  of  a  fowl. 

Form. — Short  thick  rods  forming  long  threads  coiled  up, 
which  finally  break  up  into  spores,  which  were  once  thought  to 
be  micrococci. 

Properties. — Very  motile;  does  not  dissolve  or  liquefy  gela- 
tin. 

Growth. — In  thirty  hours  abundant  growth;  aerobic;  grows 
best  at  20°  C. 

Plates. — Small  white  points  which  form  the  center  of  a  very 
fine  netting.  With  high  power  this  netting  is  found  composed 
of  bacilli  in  coils,  like  braids  of  hair. 

Excellent  impress  or  "Klatsch"  preparations  are  obtained 
from  these  colonies. 

Staining. — Ordinary  dyes. 

Bacillus  Subtilis  (Hay  Bacillus)  (Ehrenberg) .— Origin.— 
Hay  infusions;  found  also  in  air,  water,  soil,  feces,  and  putre- 
fying liquids.  Very  common,  often  contaminates  cultures. 

Form. — Large  rods,  three  times  as  long  as  broad;  slight 
roundness  of  ends;  seldom  found  singly;  usually  in  long  threads. 
Flagella  are  found  on  the  ends.  Spores  of  oval  shape,  strongly 
shining,  very  resistant. 


NON- PATHOGENIC   BACTERIA  95 

Properties. — Very  motile;   dissolves  gelatin. 

Growth. — Rapid;  strongly  aerobic. 

Plate. — Round,  gray  colonies  with  depressed  white  center. 
Under  microscope  the  center  yellow;  the  periphery  like  a  wreath, 
with  tiny  little  rays  projecting;  very  characteristic. 

Potato. — A  thick  moist  skin  forms  in  twenty-four  hours. 

Staining. — Rods,  ordinary  stain;  spores,  spore  stain. 

It  is  easily  obtained  by  covering  finely  cut  hay  with  distilled 
water,  and  boiling  a  quarter  of  an  hour.  Set  aside  forty-eight 
hours.  A  thick  scum  will  show  itself  on  the  surface,  composed 
of  the  subtilis  bacilli,  whose  spores  alone  have  survived  the  heat. 

Bacillus  Spinosus  (Luderitz). — Called  spinosus  because 
small  spine-like  processes  are  formed  by  the  colonies. 

Origin. — In  the  juices  of  the  body  of  a  mouse  and  guinea-pig 
which  were  inoculated  with  garden-earth. 

Form. — Large  rods,  straight,  some  slightly  bent,  ends 
rounded;  often  in  long  threads. 

Properties. — Large  spores,  the  bacillus  enlarging  to  allow  the 
spores  to  develop;  very  motile;  gelatin  slowly  liquefied.  A 
gas  is  formed  in  the  culture  having  an  odor  like  Swiss  cheese. 

Growth. — The  growth  occurs  at  20°  C.  temperature  only 
when  the  oxygen  is  excluded.  Very  strongly  anaerobic.  Glu- 
cose added  to  the  gelatin  (i  to  2  per  cent.)  increases  growth. 

Colonies  in  roll  cultures  and  high  stab-cultures  appear  as  little 
spheres  surrounded  by  a  zone  of  liquefied  gelatin.  In  the 
deeper  growths  thorn-like  projections  or  spines  develop,  pro- 
ceeding from  a  gray-colored  center. 

Staining. — With  ordinary  methods. 

Some  Bacteria  Found  in  Milk.— Bacillus  Acidi  Lactici 
(Huppe). — Belongs  to  the  same  group  as  the  Bacillus  coli  com- 
munis.  (See  p.  137.) 

Origin. — In  sour  milk. 

Form. — Short  thick  rods,  nearly  as  broad  as  they  are  long, 
usually  in  pairs. 

Properties. — Immotile.  Spores,  large  shining  ones.  Does 
not  liquefy  gelatin.  Breaks  up  the  sugar  of  milk  into  lactic  acid 
and  carbonic  acid  gas,  the  casein  being  thereby  precipitated. 


96  ESSENTIALS    OF    BACTERIOLOGY 

Growth. — Slow;  is  facultative  anaerobic.     Grows  at  10°  C. 

Plate  Colonies. — First  small  white  points,  which  soon  look  like 
porcelain,  glistening.  Under  microscope  the  surface  colonies 
resemble  leaves  spread  out. 

Stab-culture. — A  thick  dry  crust  with  cracks  in  it  forms  on  the 
surface  after  a  couple  of  weeks. 

Attenuation. — If  grown  through  successive  generations,  they 
lose  the  power  to  produce  fermentation.  Several  other  bacteria 
will  give  rise  to  lactic-acid  fermentation;  but  this  especial 
one  is  almost  constantly  found  and  is  very  widespread. 

In  milk  it  first  produces  acidity,  then  precipitation  of  casein, 
and,  finally,  formation  of  gases. 

A  bacillus  described  by  Grotenfeldt,  and  called  Bacterium 
acidi  lactici,  forms  alcohol  in  the  milk.  It  was  found  in  milk 
in  Bavaria. 

Boas-Oppler  Bacillus. — Also  known  as  the  Bacillus  genic- 
ulatus.  Owing  to  the  faculty  possessed  by  this  organism  of 
growing  in  the  presence  of  amounts  of  lactic  acid  sufficient  to 
check  the  development  of  all  other  lactic-acid  formers,  it  usually 
predominates  in  stomach-contents  containing  large  amounts  of 
this  substance.  The  parent  type  is  composed  of  short  rods, 
but  in  the  presence  of  considerable  amounts  of  lactic  acid  these 
change  to  a  longer  form,  which  occurs  singly  or  in  long  chains. 
It  is  stained  brown  by  Gram's  iodin  solution.  The  bacillus 
affords  confirmatory  evidence  of  the.  presence  of  a  new  growth, 
though  it  may  occur  in  benign  conditions. 

Bacillus  Butyricus  (Hiippe). — This  bacillus  causes  buty- 
ric-acid fermentation. 

Origin. — Found  in  milk. 

Form. — Short  and  long  thin  rods  with  rounded  ends;  large 
oval  spores,  seldom  forming  threads. 

Properties. — Very  motile;  liquefies  gelatin  rapidly;  produces 
gases  resembling  butyric  acid  in  odor.  In  milk  it  coagulates  the 
casein,  decomposes  it,  forming  peptones  and  ammonia,  with  a 
bitter  taste,  and  butyric-acid  fermentation.  An  alkaline  reaction. 

Growth. — Quickly,  at  35°  to  40°  C.,  with  oxygen.  Spores 
very  resistant. 


NON-PATHOGENIC    BACTERIA  97 

Colonies. — Plate. — Small  yellow  points  which  soon  run 
together,  becoming  indistinguishable. 

Stab-culture. — A  small  yellow  skin  forms  on  the  surface  with 
delicate  wrinkles;  cloudy  masses  in  the  liquefied  portion. 

Staining. — With  ordinary  stains. 

Bacillus  Amylobacter  (Van  Tiegham) ;  synonyms  Clos- 
tridium  Butyricum  (Prazmowsky) ;  Vibrion  Butyrique  of 
Pasteur  (Fig.  51). — Origin. — Found  in  putrefying  plant-infu- 
sions, in  fossils,  and  conifera  of  the  coal  period. 

Form. — Large,  thick  rods,  with  rounded  ends,  often  found  in 
chains.  A  large  glancing  spore  at  one  end,  the  bacillus  becom- 
ing spindle-shaped  in  order  to  allow  the  spore  to  grow;  hence 
the  name,  clostridium. 

Properties. — Very  motile;  gases  arise 
with  butyric  smell.  In  solutions  of  sugars, 
lactates,  and  cellulose-containing  plants 
and  vegetables,  it  gives  rise  to  decomposi- 
tions in  which  butyric  acid  is  often  formed. 
Casein  is  also  dissolved. 

Like  granulose,  a  watery  solution  of 
iodin  will  color  blue  some  portions  of  the 
bacillus;  therefore  it  has  been  called  amy- 
lobacter. 

Growth. — It  is  strongly  anaerobic,  and 
has  not  yet  been  satisfactorily  cultivated.  Fig.  51.— Bacillus 

Bacillus  Lactis  Cyanogenus  (Bac- 
terium Syncyanum)  (Huppe) . — Origin. — Found  in  blue  milk. 

Form. — Small  narrow  rods  about  three  times  longer  than  they 
are  broad;  usually  found  in  pairs.  The  ends  are  rounded. 

Properties. — They  are  very  motile;  do  not  liquefy  gelatin; 
form  spores  usually  in  one  end.  A  bluish-gray  pigment  is 
formed  outside  of  the  cell,  around  the  medium.  The  less 
alkaline  the  media,  the  deeper  the  color.  It  does  not  act  upon 
the  milk  otherwise  than  to  color  it  blue. 

Growth. — Grows    rapidly,    requiring    oxygen.    Colonies   on 
plate.     Depressed  center,  surrounded  by  ring  of  porcelain-like 
bluish  growth.     Dark-brown  appearance  under  microscope. 
7 


98  ESSENTIALS    OF    BACTERIOLOGY 

Stab-culture. — Grows  mainly  on  surface;  a  nail-like  growth. 
The  surrounding  gelatin  becomes  colored  brown. 

Potato. — The  surface  covered  with  a  dirty  blue  scum. 

Attenuation. — After  prolonged  artificial  cultivation  loses  the 
power  to  produce  pigment. 

Staining. — By  ordinary  methods. 

Bacillus  Lactis  Erythrogenes  (Bacillus  of  Red  Milk) 
(HUppe  and  Grotenf eldt) . — Origin. — Found  in  red  milk  and 
in  the  feces  of  a  child. 

Form. — Short  rods,  often  in  long  filaments,  without  spores. 

Properties. — Does  not  possess  self-movement.  Forms  a 
nauseating  odor;  liquefies  gelatin.  Produces  a  yellow  pigment 
which  can  be  seen  in  the  dark,  and  a  red  pigment  in  alkaline 
media,  away  from  the  light.  In  milk  it  produces  the  yellow 
cream  on  top  of  the  blood-red  serum,  or  fluid  in  the  center, 
and  at  the  bottom  the  precipitated  casein. 

Growth. — Grows  rapidly  in  bouillon  and  on  potatoes;  slower 
on  the  other  media.  Plates.  A  cup-like  depression  in  the 
center  of  the  colony,  with  a  pink  coloration  around  it,  the  colony 
itself  being  slightly  yellow. 

Stab-culture. — The  growth  mostly  on  surface.  The  gelatin 
afterward  colored  red  and  liquefied. 

Potato. — A  golden-yellow  pigment  formed  at  37°  C.  after  six 
days. 

Examination  of  Milk  in  Stained  Specimen. — A  drop  of 
milk  diluted  with  a  drop  of  distilled  water  is  dried  on  the  cover- 
glass  and  fixed  by  heat.  Chloroform  methylene  blue,  prepared 
by  mixing  12  to  15  drops  of  saturated  alcoholic  solution  of 
methylene  blue  with  3  or  4  c.c.  of  chloroform,  is  used  for  stain- 
ing. The  chloroform  is  then  evaporated  by  exposing  the  speci- 
men for  a  few  minutes  to  the  air.  Bacteria  blue;  rest  of  field, 
unstained. 

Another  method  is  to  mix  a  drop  of  milk  with  two  or  three 
drops  of  a  i  per  cent,  solution  of  sodium  carbonate  on  a  cover- 
glass.  Saponification  of  the  fat  occurs  on  heating  the  mixture 
to  evaporation.  The  preparation  is  then  stained  in  the  ordinary 
manner. 


NON- PATHOGENIC    BACTERIA  99 

Some  Non-pathogenic  Bacteria  Found  in  Water.— The 
bacteria  found  are  very  often  given  to  producing  pigments  or 
phosphorescence,  and  are  a  great  number;  they  frequently 
give  rise  to  foul  gases  and,  for  the  most  part,  liquefy  gelatin 
rapidly. 

Bacillus  Violaceus. — Origin. — Water. 

Form. — A  slender  rod  with  rounded  ends,  three  times  as  long 
as  it  is  broad,  often  in  threads;  middle-sized  spores. 

Properties. — Very  motile;  forms  a  violet-blue  pigment,  which 
is  soluble  in  alcohol,  and  depends  upon  oxygen  for  its  growth. 
Rapidly  liquefies  gelatin,  but  not  agar. 

Growth. — Grows  fairly  quick,  is  facultative  anaerobic. 

Cultures  on  Plate. — At  first  the  colonies  look  like  inclosed  air- 
bubbles.  Low  power  shows  irregular  masses,  with  a  center 
containing  the  pigment  and  a  hairy-like  periphery. 

Stab-culture. — Cone-like  liquefaction  containing  air,  and  the 
pigment,  in  separated  granules,  lying  toward  the  bottom. 

Stroke  Culture  on  Agar. — A  violet,  ink-like  covering  which 
remains  for  years. 

Bacillus  Coeruleus  (Smith) . — Origin. — Schuylkill  water. 

Form. — Very  thin  rods;  2.5  //  long,  0.5  /*  wide;  often  in 
threads;  spores  were  not  found. 

Properties. — Liquefies  gelatin;  produces  a  very  deep-blue 
pigment. 

Growth. — Slowly,  with  oxygen,  at  ordinary  temperature. 

Plate. — Round  colonies  on  the  surface  of  bluish  color. 

Stab-cultures. — A  cup-shaped  liquefaction  along  the  needle- 
thrust,  with  a  sparse  growth,  the  liquefied  portion  appearing 
blue. 

Fluorescent  Bacteria. — Several  kinds  present  in  water. 

Bacillus  Erythrosporus  (Eidam) . — Origin. — Drinking- 
water  and  putrefying  albuminous  solutions. 

Form. — Slender  rods,  often  in  short  threads,  with  spores  of 
oval  shape,  and  appearing  as  if  stained  with  fuchsin. 

Properties. — Motile;  does  not  dissolve  gelatin;  produces  a 
greenish,  fluorescent  pigment,  which  appears  yellow  in  reflected 
light,  but  green  on  transmitted  light. 


1OO  ESSENTIALS    OF    BACTERIOLOGY 

Growth. — Somewhat  quickly;  facultative  anaerobic;  growth 
only  at  ordinary  temperatures. 

Plates. — White  colonies,  with  greenish-yellow  fluorescence 
around  each  colony.  Under  microscope  the  periphery  appears 
radiated. 

Stab-cultures. — Good  growth  along  the  needle-thrust;  the 
whole  gelatin  gives  out  the  fluorescence. 

Bacillus  Fluorescens  Liquefaciens. — Origin. — Water  and 
from  conjunctival  sac. 

Form. — Very  fine  little  rods;  no  spores. 

Properties. — Motile;  forms  a  greenish-yellow,  fluorescent  pig- 
ment; liquefies  gelatin. 

Growth. — Rapid  at  ordinary  temperature  and  strongly  aerobic. 

Plates. — Round  colonies,  cup-shaped  depressions,  the  solid 
gelatin  that  remains  becoming  colored  with  greenish-gellow 
fluorescence. 

Stab-culture. — On  the  surface,  air-bubble  depressions;  the 
white  colonies  in  the  bottom  of  these  depressions  and  the  solid 
gelatin  around  the  inoculation  shining  with  the  fluorescence. 

Phosphorescent  Bacteria. — Are  found  usually  in  sea-water 
or  upon  objects  living  in  the  sea. 

Bacillus  Phosphorescens  Indicus  (Fischer). — Origin. — 
Tropical  waters. 

Form. — Thick  rods,  with  rounded  ends,  sometimes  forming 
long  threads. 

Properties. — Very  motile;  liquefying  gelatin  at  a  tempera- 
ture of  25°  to  30°  C.,  with  oxygen  and  a  little  moisture,  and  in 
the  dark,  a  peculiar  electric-blue  phosphorescence  develops. 

Growth. — Slowly;  must  have  oxygen;  does  not  grow  under 
10°  C.  or  over  50°  C. 

Plates. — Little  round,  gray  points,  which  under  low  power 
appear  as  green  colonies  with  reddish  tinge  around  them. 
Cooked  fish,  when  smeared  upon  the  surface  with  a  little  of  the 
culture,  show  the  phosphorescence  most  marked.  Grows  well 
on  potatoes  and  blood-serum. 

Bacillus  Phosphorescens  Indigenus  (Fischer)^.— Origin. — 
Waters  in  the  northern  part  of  Germany.  It  differs  from  the 


NON- PATHOGENIC    BACTERIA  IOI 

Indian  bacillus  in  that  it  grows  at  a  temperature  of  5°  C.,  and 
does  not  develop  upon  potatoes  or  blood-serum. 

Bacillus  Phosphorescens  Gelidus  (Forster). — Origin. — 
Surfaces  of  salt-water  fish. 

Form. — Short,  thick  rods,  looking  oval  sometimes;  zooglea 
are  often  formed. 

Properties. — Motile;  does  not  liquefy  gelatin;  a  beautiful 
phosphorescence  from  the  surface  of  fish;  it  can  be  photographed 
by  its  own  light. 

Colonies. — Grows  best  between  10°  and  20°  C.;  grows  slowly, 
and  mostly  on  the  surface.  The  material  must  contain  salt. 
A  bouillon  made  with  sea-water  or  3  to  4  per  cent,  common 
salt  will  suffice.  The  colonies  appear  as  those  of  the  Phospho- 
rescens Indicus. 

Fresh  herring  laid  between  two  plates  will  often  show  phos- 
phorescence in  twenty-four  hours. 

Three  varieties  require  glucose  in  the  culture  before  they  give 
out  any  glow.  They  are  Bacterium  Pflugeri,  Bacterium  Fischeri, 
and  Bacterium  Balticum.  They  do  not  dissolve  gelatin. 

Several  very  indistinct  species,  found  in  waters  from  factories 
and  in  some  of  the  mineral  waters,  deserve  yet  to  be  men- 
tioned. They  have  been  given  various  names  by  observers; 
and  a  new  classification  created.  Such  are  the  crenothrix, 
cladothrix,a,nd'beggiatoa,)which  belong  to  the  "higher  bacteria." 

Crenothrix  KUhniana  (Rabenhorst) . — Long  filaments 
joined  at  one  end;  little  rod-like  bodies  form  in  the  filaments, 
and  these  break  up  into  spores. 

Zooglea  are  also  formed  by  means  of  spores,  and  these  can 
become  so  thick  as  to  plug  up  pipes  and  carriers  of  water. 
They  are  not  injurious  to  health. 

Cladothrix  Dichotoma  (Cohn). — Very  common  in  dirty 
waters.  The  filaments  branch  out  at  acute  angles,  otherwise 
resembling  the  crenothrix;  accumulations  of  ocher-colored 
slime,  consisting  of  filaments  of  this  organism,  are  found  in 
springs  and  streams.  (See  Fig.  128.) 

Leptothrix  Buccalis. — In  the  mouth,  long  filaments  or 
threads  resembling  bacteria  are  commonly  found.  At  one  end 


102         ESSENTIALS  OF  BACTERIOLOGY 

are  seen  numerous  cocci-like  bodies,  which  some  regard  as 
spores.  A  variety  of  this,  or  a  nearly  allied  organism,  is  the 
most  frequent  cause  of  noma  or  gangrenous  stomatitis. 

With  iodin  the  leptothrix  is  colored  yellow.  At  one  time 
it  was  considered  the  cause  of  " tartar"  on  the  teeth,  and 
often  it  fills  the  crypts  of  the  tonsils,  forming  there  small 
masses  which  are  difficult  to  remove.  Miller  distinguishes 
three  varieties — Leptothrix  buccalis  innominata,  maxima,  and 
gigantea. 

Beggiatoa  Alba  (Vancher). — The  most  common  of  this 
species.  The  distinction  between  this  and  the  preceding  species 
lies  in  the  presence  of  sulphur  granules  contained  in  the  struc- 
ture, and  hence  they  are  often  found  where  sulphur  or  sulphids 
exist;  but  where  the  remains  of  organic  life  are  decomposing 
they  can  also  be  found. 

Several  large  spirilla  and  vibrio  live  in  bog  and  rain-water, 
but  our  space  does  not  suffice  to  describe  them.  For  the 
Bacteriologic  Examination  of  Water  see  page  220. 

Microorganisms  Found  in  Urine. — When  freshly  passed, 
urine  of  a  normal  state  contains  no  bacteria.  By  contact  with 
the  air  and  the  urinary  passages  exposed  to  air,  a  great  number 
of  yeast  molds  and  bacteria  soon  accumulate  in  the  fluid. 
Bacteria  also  enter  urine  through  the  blood  and  during  its 
secretion. 

A  number  of  bacteria  have  the  property  of  converting  urea 
into  carbonate  of  ammonia. 

The  urine  should  be  centrifuged  and  the  deposit  then  exam- 
ined. The  drying  and  fixing  must  proceed  very  slowly,  since 
otherwise  crystals  of  salts  will  be  precipitated  and  mar  the 
specimen. 

Bacterium  Ureae. — Origin. — Decomposed  ammoniacal 
urine. 

Form. — Thick,  little  rods,  with  round  ends  one-half  as  thick 
as  they  are  long. 

Properties. — Does  not  dissolve  gelatin;  changes  urea  into 
ammonium  carbonate. 

Growth. — At  ordinary  temperatures,  very  slowly.     In  two 


NON-PATHOGENIC   BACTERIA  103 

days  on  gelatin  very  minute  points,  which  in  ten  days  have  the 
size  of  a  cent.  The  colonies  grow  in  concentric  layers. 

Micrococcus  Ureae  (Pasteur  and  Van  Tiegham)  .—Origin. 
— Decomposed  urine  and  in  the  air. 

Form. — Cocci,  diplococci,  and  streptococci. 

Properties. — Decomposes  urea  into  ammonium  carbonate; 
does  not  liquefy  gelatin. 

Growth. — Grows  rapidly,  needing  oxygen;  can  remain  sta- 
tionary below  o°  C.,  growing  again  when  a  higher  temperature 
is  reached. 

Colonies  on  Plate. — On  the  surface  like  a  drop  of  wax. 

Stab-cultures. — Looks  like  a  very  delicate  thread  along  the 
needle-thrust. 

Other  bacteria  are  found  in  urine  in  various  pathologic  proc- 
esses, such  as  tubercle  bacilli,  typhoid  bacilli,  gonococci,  and 
other  pyogenic  organisms. 

The  Urobacillus  liquefaciens,  found  by  Schnitzler  and 
Krogius  in  cystitis,  is  supposed  to  stand  in  close  relationship  to 
this  disease. 

Spirilla. — A  number  of  non-pathogenic  spirilla  have  been 
described. 

Spirillum  Rubrum  (Esmarch) . — Origin. — Body  of  a  mouse 
dead  with  septicemia. 

Form. — Spirals  of  variable  length,  long  joints,  flagella  on  each 
end;  no  spores. 

Properties. — Does  not  liquefy  gelatin;  very  motile;  pro- 
duces a  wine-red  pigment,  which  develops  only  in  absence  of 
oxygen. 

Growth. — Can  grow  with  oxygen,  but  is  then  colorless;  grows 
very  slowly;  ten  to  twelve  days  before  any  sign;  grows  best  at 
37°  C. 

Gelatin  Roll-cultures. — Small,  round;  first  gray,  then  wine- 
red  colonies. 

Stab-cultures. — A  red-colored  growth  along  the  whole  line;  it 
is  deepest  below,  getting  paler  as  it  approaches  the  surface. 

Spirillum  Concentricum  (Kitasato) . — Origin. — Decom- 
posed blood. 


104  ESSENTIALS    OF    BACTERIOLOGY 

Form. — Short  spirals,  two  or  three  turns,  with  pointed  ends; 
it  has  flagella  on  the  ends. 

Properties. — Very  motile;  does  not  liquefy  gelatin. 

Growth. — Very  slow;  mostly  on  the  surface;  best  at  ordinary 
temperatures. 

Plates. — A  growth  of  rings  concentrically  arranged,  every 
alternate  one  being  transparent;  the  furthest  one  from  the 
center  possessing  small  projections. 

Stab-cultures. — Growth  mostly  on  the  surface. 

Sarcina. — Cocci  in  cubes  or  packets  of  colonies.  A  great 
number  have  been  isolated,  many  producing  very  beautiful 
pigments.  The  majority  of  them  found  in  the  air. 

Sarcina  Lutea  (Schrbter) . — Origin. — Air. 

Form. — Very  large  cocci  in  pairs;  tetrads  and  groups  of 
tetrads. 


Fig.  52. — Sarcina  ventriculi  from  stomach-contents  (x  53°)  (Van  Valzah 
and  Nisbet). 

Properties. — Liquefies  gelatin  slowly;  produces  sulphur-yel- 
low pigment. 

Growth.- — Slowly,  at  various  temperatures;  strongly  aerobic. 

Plates. — Small,  round,  yellow  colonies. 

Stab-cultures. — Grows  more  rapidly,  the  growth  being  nearly 
all  on  the  surface,  a  few  separated  colonies  following  the  needle- 
thrust  for  a  short  distance.  Agar,  a  very  beautiful  yellow, 
along  the  stroked  surface. 


PATHOGENIC   BACTERIA  105 

Sarcina  Aurantica. — Flava,  rosea,  and  alba  are  some  of  the 
other  varieties.  Many  are  obtained  from  beer. 

Sarcina  Ventriculi  (Goodsir)  (Fig.  52). — Origin. — Stomach 
of  man  and  animals. 

Form. — Colorless,  oval  cocci,  in  groups  of  eight  and  packets 
of  eight. 

Properties. — Does  not  liquefy  gelatin;  shows  the  reaction  of 
cellulose  to  iodin. 

Growth. — Rapid.  At  end  of  thirty-six  hours,  round,  yellow 
colonies,  from  which  colorless  cocci  and  cubes  are  obtained. 

Habitat. — They  are  found  in  many  diseases  of  the  stomach, 
especially  when  dilatation  exists.  Also  normally;  increased 
when  fermentation  occurs. 


CHAPTER  XVIII 
PATHOGENIC  BACTERIA 

WE  have  divided  this  part  into  two  portions: 

I.  Bacteria  which  are  pathogenic  for  man  and  other  animals. 

II.  Bacteria  which  do  not  affect  man,  but  are  pathogenic 
for  the  lower  animals. 

Here  again  it  will  be  possible  to  give  only  the  more  impor- 
tant bacteria ;  there  are  many  diseases  in  which  microorganisms 
have  been  found,  but  they  have  not  yet  been  proved  as  causa- 
tive of  the  disease,  and  have  also  been  found  in  other  diseases. 
We  cannot  treat  of  them  here. 

Bacillus  Anthracis  (Rayer  and  Davaine). — Rayer  and 
Davaine,  in  1850,  first  described  this  bacillus;  but  Pasteur,  and 
later  Koch,  gave  it  the  importance  it  now  has. 

Synonyms. — Bactericie  du  charbon  (Fr.),  Milzbrand  bacillus 
(German) ;  bacillus  of  splenic  fever  or  malignant  pustule. 

Origin. — In  blood  of  anthrax-suffering  animals. 


IO6  ESSENTIALS    OF    BACTERIOLOGY 

Form. — Rods  of  variable  length,  nearly  the  size  of  a  human 


^^ 


Fig.  53- — Bacillus  anthracis,  stained  to  show  the  spores  (X  1000)  (Frankel 
and  Pfeiffer). 


Fig.  54. — Anthrax  bacilli  in  human  blood  (fuchsin  staining)  (Zeiss  one-twelfth 
oil-immersion;  No.  4  ocular)  (taken  from  Vierordt). 

blood-corpuscle;  broad,  cup-shaped  ends;  in  bouillon  cultures 
long  threads  are  formed,  with  large  oval  spores  (Figs.  53,  54). 


PATHOGENIC    BACTERIA 


107 


Properties. — Liquefies  gelatin;  immotile;  the  spores  are  very 
resisting,  living  twenty  years,  and  resist  boiling  for  five  minutes. 

Growth. — Grows  rapidly,  between  12°  C.  and  45°  C.,  and 
requires  plenty  of  oxygen,  but  may  be  classed  as  a  facultative 
anaerobe;  grows  well  in  all  media. 

Plates  of  Gelatin. — Colonies  develop  in  two  days;  white  shiny 
spots,  which  appear  under  microscope  as  slightly  yellowish 


•--  ~~  --^Tv^— — ,^ 

--- 


Figs.  55,  56. — Stab-cultures  of  anthrax  in  gelatin. 

granular  twisted  balls,  like  a  ball  of  yarn;  each  separate  string 
or  hair,  if  looked  at  under  high  power,  being  composed  of  bac- 
teria in  threads. 

Stab-cultures. — A  white  growth  with  thorn-like  processes 
along  the  needle-track;  later  on,  gelatin  liquefied,  and  flaky 
masses  at  the  bottom.  (See  Figs.  55,  56.) 

Potato. — A  dry,  creamy  layer,  and  when  placed  in  brood-oven, 
rich  in  spores. 


I08  ESSENTIALS    OF    BACTERIOLOGY 

Varieties. — Asporogenic. — By  cultivation  in  gelatin,  contain- 
ing i  :  1000  phenol,  a  variety  develops  that  cannot  produce 
spores.  Also  involution  forms,  differing  from  the  usual  type. 

Staining. — They  readily  take  all  the  anilin  dyes  with  the 
ordinary  methods.  To  bring  out  the  cup-shaped  concave 
extremities,  a  very  weak  watery  solution  of  methylene-blue  is 
best. 

Spores  are  stained  by  the  usual  method.  When  several  bacilli 
are  joined  together,  the  place  of  their  joining  looks  like  a  spore, 
because  of  the  hollowed  ends.  The  double  staining  will  develop 
the  difference. 

Sections  of  tissue  are  stained  according  to  the  ordinary 
methods,  taking  Gram's  method  very  nicely. 

Pathogenesis. — When  mice  are  inoculated  with  anthrax  mate- 
rial through  a  wound  in  the  skin,  they  die  in  twenty-four  hours 
from  an  active  septicemia,  the  point  of  inoculation  remaining 
unchanged.  The  following  appearances  then  present  them- 
selves: 

Peritoneum. — Covered  with  a  gelatinous  exudate. 

Spleen. — Very  much  swollen,  dark  red,  and  friable. 

Liver. — Parenchymatous  degeneration. 

Blood. — Dark  red.  The  bacilli  are  found  wherever  the  capil- 
laries are  spread  out,  in  the  spleen,  liver,  intestinal  villi,  and 
glomeruli  of  kidney,  and  in  the  blood  itself.  Only  when  the 
capillaries  burst  are  they  found  in  the  tubules  of  the  kidney. 

Mode  of  Entrance. — The  bacilli  can  be  inhaled,  and  then  a 
pneumonia  is  caused,  the  pulmonary  cells  containing  the  bacilli ; 
when  the  spores  are  inhaled,  a  general  infection  occurs. 

Feeding. — The  cattle  graze  upon  the  meadows,  where  the 
blood  of  anthrax  animals  has  flowed  and  become  dried,  the 
spores  remaining,  which  then  mix  with  the  grass  and  so  enter 
the  alimentary  tract;  here  they  then  cause  the  intestinal  form 
of  the  disease,  ulcerating  through  the  villi. 

Local  Infection. — In  man  usually  only  a  local  action  occurs;  by 
reason  of  his  occupation — wool-sorter,  cattle-driver,  etc. — he 
obtains  a  small  wound  on  the  hand,  and  local  gangrene  and 
necrosis  set  in,  but  death  follows  in  the  severer  forms  from  a 


PATHOGENIC   BACTERIA  109 

general  pyemia;  there  is  severe  edema  of  the  tissues  in  and 
about  the  wound,  and  a  pulmonary  edema  likewise  'occurs. 
Wounds  about  the  face  and  neck  are  more  fatal. 

Pneumonia  by  inhalation  and  intestinal  infection  also  occurs 
in  man. 

Susceptibility  of  Animals, — Dogs,  birds,  and  cold-blooded 
animals  affected  the  least;  while  mice,  sheep,  and  guinea-pigs 
quickly  and  surely. 

Products  of  Anthrax  Bacilli. — A  basic  ptomain  has  not  been 
found,  but  a  toxalbumin  or  proteid,  called  anthraxin,  has  been 
obtained.  A  certain  amount  of  acid  is  produced  by  the  virulent 
form,  alkali  by  the  weak. 

Attenuation  and  Immunity. — Cultures  left  several  days  at  a 
temperature  between  40°  and  42°  C.  soon  become  innocuous, 
and  when  injected  into  animals  protect  them  against  the  viru- 
lent form. 

The  lymph  obtained  from  lymph-sac  of  a  frog  destroys  the 
virulence  of  anthrax  bacilli  and  spores  temporarily. 

Hankin  obtained  an  alexin  from  the  blood  and  spleen  of  rats, 
they  being  naturally  immune.  It  destroyed  the  anthrax  bacilli 
in  vitro,  and  used  by  injection  in  susceptible  animals,  made 
them  immune.  It  is  insoluble  in  alcohol  or  water. 

Protective  Vaccination. — Animals  have  been  rendered  immune 
by  various  ways — by  inoculation  of  successive  attenuated  cul- 
tures; also  with  sterilized  cultures — that  is,  cultures  containing 
no  bacilli,  and  with  cultures  of  other  bacteria. 

Habitat. — In  the  serum  about  the  wound  and  in  the  blood 
anthrax  bacilli  are  readily  found.  Tannery  employes  are 
frequent  sufferers. 

The  bacillus  has  never  been  found  free  in  nature. 

Bacillus  Tuberculosis  (Koch). — This  very  important 
bacillus  was  first  described,  demonstrated,  and  cultivated  by 
Koch,  who  made  his  investigations  public  on  the  24th  of  March, 
before  the  Physiological  Society  of  Berlin,  in  the  year  1882. 

Origin. — In  various  tuberculous  products  of  man  and  other 
animals. 

Form. — Very  slender  rods,  nearly  straight,  about  one-quarter 


IIO         ESSENTIALS  OF  BACTERIOLOGY 

the  size  of  a  red  corpuscle's  diameter,  their  ends  rounded, 
usually  solitary,  often,  however,  lying  in  pairs  in  such  a  manner 
as  to  form  an  acute  angle.  Sometimes  they  are  S-shaped.  In 
colored  preparations  little  oval  spaces  are  seen  in  the  rod,  which 
resemble  spores;  but  the  question  of  the  existence  of  spores  is 
still  undecided.  (See  Figs.  57,  58.) 
Properties. — Does  not  possess  self-movement. 


X- 
••»•. 


I  ' 

A  -     ^ 


Fig.  57. — Tubercle   bacilli   in   sputum;    carbol-fuchsin   and  methylene-blue 
(Zeiss  one-twelfth  oil-immersion). 


Growth. — Requires  special  media  for  its  growth,  and  a  temper- 
ature varying  but  slightly  from  37-5°C.  It  grows  slowly, 
developing  first  after  ten  days,  reaching  its  maximum  in  three 
weeks.  It  is  facultative  anaerobic.  On  gelatin  it  does  not 
form  a  growth. 

Colonies  on  Blood-serum. — Koch  first  used  blood-serum  for 
culture,  and  obtained  thereon  very  good  growths.  Test-tubes 
with  stroke  culture  are  placed  in  the  brood-oven  at  37°  C. 
for  ten  to  fourteen  days,  when  small  glistening  white  points 
appear,  which  then  coalesce  to  form  a  dry,  white,  scale-like 


PATHOGENIC   BACTERIA  III 

growth.  Under  microscope  composed  of  many  fine  lines  con- 
taining the  tubercle  bacillus. 

Glycerin-agar. — By  adding  4  to  6  per  cent,  glycerin  to 
ordinary  agar-peptone  medium,  Nocard  and  Roux  obtained  a 
culture-media  upon  which  tubercle  bacilli  grow  much  better 
than  upon  blood-serum.  This  is  now  almost  exclusively  used. 

Stroke  cultures  are  here  used  as  with  blood-serum.  They  are 
placed  in  incubator  after  inoculation,  and  remain  there  about 
ten  days,  at  a  temperature  of  37°  C.  The  cotton  plugs  of  the 
tubes  are  covered  with  rubber  caps,  the  cotton  first  having  been 


Fig.  58. — Giant-cell  containing  bacilli  (from  a  photograph  made  by  Dr.  Wm. 
M.  Gray). 

passed  through  the  flame,  and  moistened  with  a  few  drops 
of  sublimate  solution.  The  rubber  cap  prevents  the  evaporation 
of  the  water  of  condensation,  which  always  forms  and  keeps 
the  culture  from  drying  up. 

The  growth  which  occurs  resembles  the  rugae  of  the  stomach, 
and  sometimes  looks  like  moistened  crumbs  of  bread.  The 
impression  or  "Klatsch"  preparation  shows  under  the  micro- 
scope a  thick,  curled-up  center  around  which  threads  are 
wound  in  all  directions.  And  these  fine  lines  show  the  bacilli 
in  profusion. 

Potato. — It  can  be  cultivated  on  slices  of  potato  which  are 
placed  in  air-tight  test-tubes. 


112 


ESSENTIALS    OF    BACTERIOLOGY 


Bouillon.  —  Bouillon  containing  4  per  cent,  glycerin  is  a  very 
good  nurture  ground. 

Varieties.  —  Branching  and  other  aberrant  forms  are  not  rare, 
and  the  tendency  now  is  to  class  the  organism  with  the  "higher 
bacteria."  Other  acid-fast  bacilli  exhibit  similar  types,  and  it  is 
possible  that  the  bacillary  parasitic  form  is  only  one  stage  in 
the  life-history  of  the  organism. 

Little  granules,  arranged  like  streptococci,  which  take  the 
characteristic  stain,  and  look  as  if  the  protoplasm  had  been 


'-~        </  >   -£*\         \ 

<&  /^A.t.ti  ^ 

J^.<  ,/4  ./  ^j//  x     v*K> 

i^'^^'i^^N* 

i  jK          /  >*      -*•      *m      *  4*  J*?         i» 

>j^<; 


*^  / 


Fig.  59. — Tubercle  bacillus  in  sputum  (Frankel  and  Pfeiffer). 

destroyed  that  inclosed  them,  are  frequently  found  in  sputum. 

Bovine  tubercle  bacilli  are  about  one-third  smaller  than  human 
tubercle  bacilli. 

Staining. — The  tubercle  bacilli  require  special  methods  to 
stain  them,  and  a  great  number  have  been  introduced.  They 
are  stained  with  great  difficulty:  but  once  stained,  they  are 
very  resistant  to  decolorizing  agents.  Upon  these  facts  all  the 
methods  are  founded. 

It  will  be  necessary  to  describe  only  those  methods  principally 
in  use;  and  as  the  examination  of  sputum  for  bacilli  is  of  so 


PATHOGENIC    BACTERIA 


frequent  an  occurrence,  and  so  necessary,  it  is  well  to  detail  in 
particular  the  method  of  staining. 

Starting  with  the  sputum,  we  search  for  little  clumps  or  rolled- 
up  masses;  if  these  are  not  present,  the  most  solid  portions  of  the 
mucus  are  brought  with  forceps  upon  a  clean  cover-glass;  very 
little  suffices.  With  another  cover-glass  it  is  pressed  and  spread 
out  evenly;  drawing  one  glass  over  the  other,  we  obtain  two 
specimens,  and  these  are  put  aside  or 
held  high  over  the  flame  until  dry. 

If  we  desire  to  examine  the  speci- 
men quickly,  or  make  a  hurried  diag- 
nosis, we  use  the  rapid  method,  with 
hot  solutions;  otherwise  we  let  it  stay 
in  cold  solution  until  the  next  morn- 
ing, the  advantages  of  which  will  be 
later  on  described. 

The  Rapid  Method  (B.  Franker s 
Method,  Modified  by  Gobbet}. — The 
principle  is  to  combine  with  the  con- 
trast stain  the  decolorizing  agent;  but 
the  preparations  are  not  permanent; 
the  method,  however,  is  very  useful. 

Two  solutions  are  required:  one 
of  Ziehl's  carbol-fuchsin;  the  other 
Gabbet's  acid  methylene-blue.  (See 
Formula  No.  x,  on  p.  33.) 

The  cover-glass  containing  the 
dried  sputum  is  passed  three  times 
through  the  flame,  as  described  in 
the  general  directions.  It  is  then  placed  in  the  carbol-fuchsin 
solution  five  minutes  (cold) ,  or  two  minutes  in  the  hot,  immedi- 
ately then  transferred  to  the  second  solution,  the  acid  blue, 
where  it  remains  one  minute,  then  washing  in  water.  The 
preparation  is  dried  between  filter-paper,  and  mounted  best 
first  in  water.  Examined  with  oil-immersion. 

A  somewhat  longer,  but  preferable,  method  is  to  decolorize 
the  carbol-fuchsin  with  weaker  acid.     The  smear  is  treated  with 
8 


Fig.  60. — Growth  on  agar. 


114  ESSENTIALS    OF    BACTERIOLOGY 

5  per  cent,  nitric  or  10  per  cent,  sulphuric  acid  until,  after 
washing  with  water,  a  bright  pink  remains.  The  excess  of 
color  is  then  washed  out  with  95  per  cent,  alcohol  until  no 
further  color  is  imparted  to  the  alcohol,  and  the  smear  is  a 
pinkish  gray.  The  preparation  is  then  washed  with  water  and 
counterstained  with  aqueous  methylene-blue  for  ten  to  thirty 
seconds.  A  mechanical  stage  is  of  great  assistance  in  the 
search  for  the  bacilli,  as  it  permits  every  portion  of  the  prepa- 
ration to  be  inspected  systematically. 

Slow  Method. — The  stain  may  also  be  used  without  heating, 
though  in  this  case  a  much  longer  time  is  required  before  the 
bacilli  take  up  the  stain.  The  preparation  is  left  in  a  small 
dish  or  beaker  full  of  carbol-fuchsin  for  eight  to  ten  hours,  and 
then  decolorized  and  counterstained  in  the  usual  way.  The 
method  is  less  liable  to  produce  artefacts  than  the  quick 
method,  but  is  not  much  used  on  account  of  the  time  it 
takes. 

Examination  in  Urine. — In  urine,  owing  to  the  almost  inevit- 
able contamination  with  the  smegma  bacillus,  special  methods 
are  necessary  to  avoid  error.  The  preparation  may  be  left  in 
97  per  cent,  alcohol  for  eight  hours,  when  the  smegma  bacillus 
will  have  become  decolorized,  or  Pappenheim's  method  may  be 
used:  (i)  Smear  and  fix  as  usual;  (2)  stain  with  hot  carbol-fuch- 
sin for  two  minutes,  pour  off  the  surplus  dye  without  washing; 
(3)  counterstain  and  decolorize  by  pouring  five  times  over  the 
preparation  the  following  solution:  A  i  per  cent,  alcoholic  solu- 
tion of  corallin  is  saturated  with  methylene-blue  and  20  parts 
of  glycerin  added.  Wash  in  water,  dry  with  blotting-paper, 
then  in  the  air,  and  examine.  The  tubercle  bacilli  are  stained 
red,  smegma  bacilli,  blue. 

Examination  of  Milk  for  Tubercle  Bacilli.— Place  a  drop 
of  the  sample  on  a  cover-glass  and  mix  it  with  two  drops  of  a  i 
per  cent,  solution  of  sodium  carbonate.  The  cover-glass  is 
then  gently  warmed  until  evaporation  is  complete.  The 
saponified  fat  is  then  stained,  as  the  ordinary  cover-glass  prepa- 
ration. Only  a  very  few  persons  have  succeeded  in  discovering 
the  bacillus  in  milk. 


PATHOGENIC    BACTERIA  1 15 

The  bacillus  of  leprosy  resembles  the  tubercle  bacillus  in  its 
staining  properties,  but  gives  up  the  carbol-fuchsin  more  easily 
and  is  usually  decolorized  by  the  acid  and  alcohol.  It  is  colored 
blue  by  Pappenheim's  method. 

Acid-fast  bacilli  have  also  been  obtained  from  timothy  grass, 
butter,  milk,  manure,  and  the  surfaces  of  animal  bodies,  but 
differ  from  the  tubercle  bacillus  in  cultural  characteristics. 

Biedert's  Method  of  Collecting  Bacilli. — When  the  bacilli  are 
very  few  in  a  great  quantity  of  fluid,  as  urine,  pus,  abundant 
mucus,  etc.,  Biedert  advises  to  mix  15  c.c.  of  the  fluid  with 
75  to  100  c.c.  water  and  a  few  drops  of  potassium  or  sodium 
hydroxid,  then  boiling  until  the  solution  is  quite  thin.  It  is 
placed  in  a  conical  glass  for  two  days,  and  bacilli  with  other 
morphologic  elements  sink  to  the  bottom  of  the  glass;  when 
the  supernatant  liquid  is  decanted,  the  residue  can  be  easily 
examined.  In  this  way  bacilli  were  found  that  had  eluded 
detection  examined  in  the  ordinary  manner. 

The  centrifugal  machine  is  used  either  in  connection  with 
Biedert's  sediment  method  or  without,  to  obtain  the  solids  sus- 
pended in  urine  or  serum. 

When  the  bacilli  are  so  few  in  number  in  sputum  or  urine  as 
to  make  their  detection  difficult,  and  also  when  doubt  exists  as 
to  the  identity  of  acid-fast  bacilli  found,  several  guinea-pigs 
should  be  injected  in  the  groin  and  smears  and  sections  made 
from  the  enlarged  glands  resulting. 

Phenol  to  Sediment  Sputum. — Pure  phenol  added  to  sputum 
(about  i  part  of  the  acid  to  6  parts  of  sputum)  will  in  a  few 
hours  produce  a  coagulation  and  allow  the  sputum  to  be  spread 
evenly  on  the  cover-glass,  showing  greater  collections  of  bacilli. 

Without  Cover-glass. — Sputum  can  be  spread  and  stained  on 
the  glass  slide  without  the  use  of  a  cover-glass,  the  oil  of  cedar 
being  placed  directly  on  the  stained  sputum,  and  the  oil-immer- 
sion lens  dipping  into  it.  It  is  a  rapid  and  cheap  way;  and 
when  a  given  case  is  to  be  studied  daily,  the  method  is  useful. 

Pure  Cultures  from  Sputum. — Kitasato  recommends  the 
thorough  washing,  changing  the  water  ten  times,  of  the  small 
masses  found  in  the  sputum  of  tuberculous  persons.  When 


Il6  ESSENTIALS    OF    BACTERIOLOGY 

such  specimens  are  examined,  they  show  tubercle  bacilli  alone, 
and  when  inoculated  in  agar,  give  rise  to  pure  cultures. 

Staining  Bacillus  Tuberculosis  in  Tissue  (Sections). — The 
general  method  of  Gram  can  be  used,  but  the  better  way  is  to  use 
the  following: 

Carbol-fuchsin,  fifteen  to  thirty  minutes. 
5  per  cent,  sulphuric  acid,  one  minute. 
Alcohol,  until  a  light-red  tinge  appears. 
Weak  methylene-blue,  three  to  five  minutes. 
Alcohol,  for  a  few  seconds. 
Oil  of  cloves,  until  cleared. 
Canada  balsam,  to  mount  in. 

Instead  of  carbol-fuchsin,  alcoholic  solution  of  fuchsin  or 
anilin-water  fuchsin  can  be  used,  but  the  sections  must  remain 
in  the  stain  overnight. 

Hardened  Sputum  and  Sectioning. — Sputum  can  be  hardened 
by  placing  it  in  98  per  cent,  alcohol.  Thin  sections  can  be 
obtained  by  imbedding  the  hardened  sputum  in  collodion.  The 
sections  are  then  stained  as  ordinary  tissue  sections. 

To  Preserve  Sputum. — Sputum  can  be  preserved  for  future  use 
by  placing  it  in  alcohol,  where  it  can  be  kept  for  months. 
Cover-glass  preparations  can  then  be  made  by  softening  the 
coagula  with  a  small  amount  of  liquor  potassa. 

The  resisting  action  of  the  bacillus  to  acids  is  supposed 
to  be  due  to  a  peculiar  arrangement  of  the  albumin  and 
cellulose  of  the  cell,  rather  than  to  any  particular  capsule 
around  it. 

Pathogenesis. — When  a  guinea-pig  has  injected  into  its  peri- 
toneal cavity  some  of  the  diluted  sputum  containing  tubercle 
bacilli,  it  perishes  in  about  three  weeks,  and  the  following 
picture  presents  itself  at  the  autopsy:  at  the  point  of  inoculation 
there  is  a  local  tuberculosis,  little  tuberculous  nodules  contain- 
ing the  characteristic  bacilli.  In  the  lungs  and  the  lymphatics, 
similar  tubercles  are  found — a  general  tuberculosis. 

If  the  animal  lingers  a  few  weeks  longer,  the  tubercles 
becomes  necrosed  in  the  center  and  degeneration  occurs,  the 


PATHOGENIC   BACTERIA  1IJ 

periphery  still  containing  active  bacilli,  cavities  having  formed 
in  the  center. 

Since  the  bacilli  die  in  course  of  time,  killed  by  their  own 
products,  their  number  forms  no  correct  guide  of  the  dam- 
age present:  even  their  absence  in  the  sputum  does  not  preclude 
the  absence  of  a  tuberculous  process.  //  is  their  presence  only 
that  warrants  a  positive  declaration.  The  number  of  bacilli  in 
a  given  specimen  is  no  indication  of  the  severity  of  the  disease. 

They  are  found  in  the  blood  only  when  a  vessel  has  come  in 
direct  contact  with  a  tuberculous  process  through  rupture  or 
otherwise.  They  have  been  found  in  other  secretions — milk, 
urine,  etc. 

Man  is  infected  as  follows: 

Through  Wounds. — Local  tuberculosis. 

Through  Nutrition. — Milk  and  meat  of  tuberculous  animals. 
Phthisical  patients  swallowing  their  own  sputum  and  causing 
an  intestinal  tuberculosis. 

Inhalation. — This  is  the  most  usual  way,  probably  constitut- 
ing the  cause  in  nine-tenths  of  the  cases,  except  in  children. 

The  sputum  of  phthisical  patients  expectorated  on  the  floors 
of  dwelling-houses,  in  handkerchiefs,  etc.,  dries,  and  the  bacilli 
set  free  are  placed  in  motion  by  the  wind,  or  rising  with  the  dust, 
are  thus  inhaled  by  those  present.  When  the  sputum  is  kept 
from  drying  by  expectoration  in  vessels  containing  water,  this 
great  danger  can  be  avoided. 

Nearly  all  the  cases  of  heredity  can  be  explained  in  this  man- 
ner; the  young  children,  possessing  very  little  resistance,  are 
constantly  exposed  to  the  infection  through  inhalation,  and  are 
especially  prone  to  intestinal  infection  through  milk  and  other 
foods. 

Immunity. — No  one  can  be  said  to  be  immune,  though  per- 
sons who  have  been  greatly  weakened  offer  less  resistance  than 
healthy  individuals. 

Tuberculosis  in  Animals. — Tuberculosis  is  probably  the  most 
widely  disseminated  disease  among  domestic  animals,  and 
affects  cattle,  pigs,  horses,  dogs,  cats,  the  smaller  ruminants, 
birds,  and  even  turtles  and  fish.  The  conclusion  of  Koch, 


Il8         ESSENTIALS  OF  BACTERIOLOGY 

made  public  in  his  address  to  the  Tuberculosis  Congress  in 
1 901,  that  human  and  bovine  tuberculosis  are  distinct  and  that 
infection  of  human  beings  from  cattle  occurs  so  seldom  that  no 
general  regulations  to  restrict  it  are  necessary,  has  found  few 
adherents.  It  is  true  that  certain  differences  exist  between 
human  and  bovine  tubercle  bacilli,  the  latter  appearing  to  be 
more  virulent  to  animals,  and  it  is  a  fact  that  cattle  are  very 
slightly  susceptible  to  the  human  bacillus,  but  it  is  not  likely 
that  the  converse  is  so.  Children  are  particularly  liable  to 
infection  through  the  gastro-intestinal  tract,  and  it  has  been 
shown  that  the  uninjured  mucosa  of  the  infant's  intestine  is 
permeable  to  bacilli,  so  that  the  pulmonary  disease  in  the 
young  may  often  be  the  result  of  tuberculous  bronchial  nodes 
secondary  to  tuberculous  glands  of  the  mesentery. 

Various  observations  on  animals  have  shown  that  the  bacillus 
occurring  in  each  species  has  acquired  certain  special  charac- 
teristics regarding  growth  and  virulence.  The  bacilli  causing 
tuberculosis  in  the  cold-blooded  animals  have  departed  farthest 
from  the  human  type,  those  of  birds  to  a  less  degree,  and  those 
of  cattle  least  of  all. 

Products  of  Tubercle  Bacilli.— The  true  nature  of  the 
tubercle  toxin  is  not  yet  clear.  It  is  not  unlikely  that  several 
toxic  bodies  differing  from  one  another  in  their  properties  are 
produced.  Koch's  tuberculin  (1890)  was  obtained  by  filtering, 
through  unglazed  porcelain,  concentrated  glycerin  bouillon 
cultures  of  tubercle  bacilli.  It  was  speedily  shown  to  be  devoid 
of  curative  power,  and  is  now  used  mainly  for  diagnosing  the 
disease  in  cattle.  In  healthy  animals  little  or  no  reaction  is 
produced  by  the  injection  of  30  to  40  eg.  of  tuberculin,  but  if 
tuberculous,  the  temperature  rises  2°  or  3°  F.  in  eight  to  twelve 
hours,  and  remains  elevated  for  a  like  period  of  time. 

Tuberculocidin. — This  is  an  albuminoid  obtained  from  the 
original  tuberculin  by  precipitation  with  alcohol.  Klebs  used 
it  as  a  cure  for  tuberculosis. 

Tuberculin  R  is  an  extract  made  from  dried  and  powdered 
living  bacilli,  and  was  recommended  by  Koch  in  place  of  the 
original  tuberculin. 


PATHOGENIC    BACTERIA  1 19 

Bacillen  emulsion  (B.  E.)  is  similar  to  tuberculin  R,  and 
is  a  glycerin  emulsion  of  crushed  bacteria.  Theo.  Smith 
recommends  virulent  uncrushed  bacteria  killed  by  moderate 
heat. 

Denys'  B.  F.  tuberculin  is  a  nitrate  of  liquid  cultures  to 
which  0.25  per  cent,  phenol  has  been  added  and  allowed  to 
stand  two  weeks.  It  is  prepared  in  eight  dilutions. 

Opsonic  Treatment. — In  recent  years  the  use  of  tuberculin  R 
has  again  been  brought  forward  by  Wright  and  others  and 
curative  claims  made  for  it.  It  is  used  in  very  small  doses, 
T-Jau  milligram  at  intervals  of  several  days,  and  the  effect  on 
the  opsonic  index  carefully  watched. 

Use  of  Tuberculin. — In  the  use  of  tuberculin  severe  reactions 
are  to  be  avoided.  The  smallest  dose  possible  is  commenced 
with.  Trudeau  uses  for  afebrile  cases  YO^OOO  milligram  liquid 
measure  Koch's  B.  E.,  or  Denys'  B.  F.,  increasing  i  decigram 
every  three  days  until  i  c.c.  can  be  injected  without  causing 
any  reaction.  This  treatment  must  extend  over  months. 
Tuberculin  immunity  does  not  last  indefinitely. 

Ophthalmic  Tuberculin  Reaction  of  Calmette. — A  modified 
form  of  tuberculin  is  placed  on  the  conjunctiva  of  an  individual 
suspected  of  having  tuberculosis.  In  a  few  hours  a  congestion, 
more  or  less  severe,  results,  and  lasts  several  days.  In  healthy 
persons  no  reaction  occurs.  The  test  is  claimed  to  be  harmless, 
though  severe  reactions  have  been  reported,  in  tuberculous 
patients,  and  even  in  healthy  persons  a  second  application  to 
the  same  eye  may  cause  an  inflammatory  reaction. 

Agglutination. — Arloing  and  Courmont  have  described  an 
agglutination  reaction  for  the  tubercle  bacillus  similar  to  the 
Widal  reaction  of  typhoid  fever  (see  page  131).  It  is  very 
unreliable,  however,  and  but  little  importance  is  attached  to  it. 

Antituberculous  Serum. — The  attempts  to  produce  an 
effective  serum  have  so  far  been  unsuccessful.  Marmorek, 
by  growing  the  bacillus  on  a  special  serum  obtained  by  injecting 
calves  with  the  leukocytes  of  guinea-pigs,  has  secured  a  toxin 
which  he  used  to  immunize  horses,  and  the  serum  so  obtained 
has  been  tried  with  encouraging  results,  but  its  value  is  still 


120  ESSENTIALS    OF    BACTERIOLOGY 

doubtful.  Several  other  serums  have  been  introduced,  but 
none  have  shown  any  lasting  virtues. 

Lepra  Bacillus  (Hansen). — Origin. — In  1880  Armauer 
Hansen  declared,  as  the  result  of  many  years'  investigation, 
that  he  found  a  bacillus  in  all  leprous  processes. 

Form. — Small  slender  rods,  somewhat  shorter  than  tubercle 
bacilli,  otherwise  very  similar  in  appearance. 

In  the  interior  of  the  cell  two  or  three  oval  spaces  are  usually 
seen,  not  known  if  spores  or  otherwise. 

Properties. — They  are  immotile,  and  do  not  liquefy  the 
nutrient  media. 

Growth. — Bordoni-Uffreduzzi  have  obtained  growths  upon 
blood-serum  to  which  peptone  and  glycerin  had  been  added, 
but  the  accuracy  of  this  observation  is  very  doubtful. 

Staining. — They  resist  the  decolorizing  action  of  acids,  as  the 
tubercle  bacilli,  but  they  are  easily  stained,  requiring  but  a 
few  minutes  with  the  ordinary  watery  solutions.  They  take 
Gram's  stain  readily. 

Patho  genesis. — Arning  has  inoculated  prisoners  with  tissue 
obtained  from  leprous  patients,  and  produced  true  leprosy. 

Rabbits  which  had  been  infected  through  the  anterior  cham- 
ber of  the  eye  showed  the  lepra  nodules  (containing  the  lepra 
bacilli)  diffused  through  various  organs,  but  here  again  the 
.results  are  not  wholly  satisfactory. 

In  man  the  skin  and  peripheral  nerves  are  principally  affected, 
but  the  lymphatic  glands,  liver,  and  spleen  can  also  become  the 
seat  of  the  lepra  nodules.  The  lepra  cells  which  compose  these 
nodules  contain  the  bacilli  in  large  numbers.  By  applying  a 
vesicant  to  the  leprous  skin,  the  serum  thereby  obtained  will 
contain  great  numbers  of  bacilli.  This  is  a  simple  diagnostic 
test. 

Method  of  Infection. — Not  yet  determined;  the  air,  soil,  water, 
and  food  of  leprous  districts  have  been  carefully  examined  with- 
out result.  The  nasal  secretion  is  very  infectious. 

Syphilis  Bacillus  of  Lustgarten  (Smegma  Bacillus  of 
Alvarey  and  Tavel). — Lustgarten,  in  1885,  through  a  certain 
staining  process,  found  peculiar  bacilli  in  syphilitic  tissues 


PATHOGENIC   BACTERIA 


121 


which  he  thought  had  a  direct  connection  with  the  disease. 
But  this  has  been  disproved  and  the  cause  has  been  found 
in  a  protozoon  which  has  been  called  Spirochaeta  pallida, 
which  see  (p.  194). 

Bacillus  of  Glanders  (Bacillus  Mallei  (Lbffler-ShUtz) ; 
Rotz-bacillus) .— Origin.— In  the  "farcy  buds"  or  little 
nodules  of  the  disease,  by  Loffler  and  Shiitz,  in  1882. 

Form. — Small  slender  rods,  about  the  size  of  the  tubercle 
bacillus.  The  ends  rounded.  Never  appearing  in  large  collec- 


Fig.  61. — Bacillus  of  glanders  from  a  culture  upon  glycerin  agar-agar  (x  1000) 
(Frlinkel  and  Pfeiffer). 

tions,  usually  singly.  Spores  are  said  to  exist,  but  this  is  doubt- 
ful (Fig.  61). 

Properties. — The  rods  are  very  resistant,  living  in  a  dried 
state  for  three  months  and  longer  without  any  spores  present. 
They  are  not  motile;  possess,  however,  great  molecular  vibra- 
tion. 

Growth. — The  growth  occurs  between  25°  and  40°  C. — best 
at  37°  C.;  it  is  very  sparse  upon  gelatin,  but  on  glycerin-agar 
or  blood  serum  a  very  abundant  growth  occurs. 


122  ESSENTIALS    OF    BACTERIOLOGY 

Colonies. — On  agar  or  glycerin-agar  there  appear  in  two  to 
three  days  small  white  glistening  drops,  which  under  microscope 
seem  as  round  granular  masses  with  an  even  periphery. 

Stroke  Cultures. — On  glycerin-agar  and  blood-serum  small 
transparent  drops  of  whitish  or  grayish  color,  which  soon 
coalesce  to  form  a  broad  band. 

Potato. — An  amber-colored,  honey-like  growth  which  grad- 
ually turns  red,  then  brown,  and  greenish-brown  around  it. 
Weakly  acid  potatoes  are  a  good  medium  and  give  the  most 
typical  growth. 

Staining. — Since  the  bacillus  is  very  easily  decolorized,  some 
special  methods  have  been  recommended. 

Loffler's  (jor  cover-glass  preparations): 

1.  Alkaline  methylene-blue  (Loffler's),  five  minutes. 

2.  Acetic  acid  with  a  few  drops  of  tropaeolin,  one  second. 

3.  Washed  in  water. 

For  Sections. — Instead  of  tropaeolin  acetic  acid,  the  following 
mixture  is  used: 

Oxalic  acid  (5  per  cent.) i  drop. 

Concentrated  sulphuric  acid 2  drops. 

Distilled  water 2  drams.— M. 

The  sections  are  kept  in  this  five  seconds. 
Kuhne's  method  (cover-glass): 

1.  Warm  carbol-blue,   two  minutes. 

2.  Decolorized  in  weak  solution  of  muriatic  acid  (10  :  500). 

3.  Washed  in  water. 
Sections  of  tissue: 

1.  Carbol-blue,  one-half  hour. 

2.  Decolorized  in  J  per  cent,  muriatic  acid. 

3.  Washed  in  distilled  water. 

4.  Dehydrated  in  alcohol  one  second. 

5.  Anilin-oil  with  6  drops  of  turpentine,  five  minutes. 

6.  Turpentine,  xylol,  Canada  balsam. 

If  contrast  stain,  add  5  drops  of  safranin  (Bismarck-brown) 
to  turpentine,  and  use  it  after  the  xylol. 


PATHOGENIC   BACTERIA  123 

Pathogenesis. — If  horses,  field-mice,  or  guinea-pigs  be 
inoculated  subcutaneously,  with  but  a  very  small  quantity  of 
culture,  a  local  affection  results,  followed  some  time  after  by 
a  general  disturbance;  ulcers  form  at  the  point  of  inoculation; 
little  nodules,  which  then  caseate,  leaving  scars  and  involving 
the  lymphatics;  metastatic  abscesses  then  occur  in  the  spleen 
and  lungs,  and  death  arises  from  exhaustion.  Cattle,  pigs, 
and  rabbits  are  not  easily  affected;  man  is  readily  attacked. 
The  bacilli  gain  entrance  to  the  blood  and  urine.  Nasal 
glanders  occurs  whatever  the  mode  of  inoculation. 

Manner  of  Infection. — Glanders  being  a  highly  contagious 
disease,  it  requires  but  a  slight  wound  to  allow  it  to  gain 
entrance. 

In  horses  the  primary  sore  seems  to  be  at  the  nasal  mucous 
membrane.  In  man  it  is  usually  on  the  fingers.  Boiling  water 
or  i  :  10,000  sublimate  solution  will  quickly  destroy  the  viru- 
lence of  this  bacillus. 

Mallein. — A  substance  called  mallein  has  been  obtained 
from  the  cultures  grown  in  glycerin  bouillon.  It  gives  a  reaction 
when  injected' into  cattle  suffering  from  glanders,  and  is  said 
to  be  useful  in  diagnosing  the  disease. 

Bacillus  of  Diphtheria  (Klebs-Lbffler).— Origin.— Klebs 
found  it  in  membrane  in  1883;  it  was  isolated  by  Loffler  in 
1884. 

Form. — Small,  slightly  curved  rods  about  as  long  as  tubercle 
•bacilli  and  twice  as  broad;  the  ends  are  at  times  swollen; 
spores  have  not  been  found.  Their  form  is,  however,  very 
variable — sometimes  much  longer  than  usual,  one  end  often 
greatly  knobbed.  Normal  bacilli  are  found  only  in  mem- 
brane. 

Stained  forms  are  characteristic,  since  the  ends  are  more  easily 
colored  than  the  center,  and  usually  the  bacillus  stains  in  seg- 
ments, so  that  it  seems  to  be  made  up  of  very  short  sections. 
At  first  sight  it  appears  like  a  chain  of  cocci. 

Small  granules,  the  Babes-Ernst  granules,  are  shown  by  the 
special  staining  of  Neisser. 

Properties. — They  do  not  possess  any  movement;  do  not 


124  ESSENTIALS    OF    BACTERIOLOGY 

liquefy  gelatin.  They  are  not  very  resistant,  being  destroyed 
by  a  temperature  of  50°  C.,  but  they  have  lived  on  blood-serum 
five  months.  Acid  is  produced  in  sugar  media. 

Growth. — Grow  readily  on  all  media,  but  best  on  blood-serum 
mixtures,  between  temperatures  of  20°  and  40°  C.  They  are 
facultative  anaerobic;  they  grow  quite  rapidly  and  profusely. 
Egg  cultures  (Hueppe's  method)  give  good  growths.  Passing 
currents  of  air  increase  the  growth. 


Fig.  62. — Diphtheria  bacilli  from  a  culture  on  blood-serum,  stained  by 
Loffler's  methylene-blue  solution,  showing  deeply  stained  points;  (x  2000) 
(Wright  and  Brown). 

Colonies  on  Gelatin  Plates. — At  24°  C.  little  round  colonies, 
white  under  low  power,  granular  center;  irregular  borders. 

Stab-cultures. — Small,  white  drops  along  the  needle-track.  In 
glycerin-agar  a  somewhat  profuse  growth. 

Potato. — On  alkaline  surface,  a  grayish  layer  in  forty-eight 
hours. 

Blood-serum  (After  Lbffler) . — Blood-serum  3  parts,  and  bouil- 
lon i  part;  the  bouillon  contains  peptone,  i  per  cent.,  sodium 
chlorid,  \  per  cent.,  and  dextrin  (or  glucose),  i  per  cent. 

In  a  few  hours  (eight  to  sixteen)  on  the  white  opaque  surface 
a  slight  moisture  is  noticeable  which,  if  examined,  is  composed 


PATHOGENIC   BACTERIA 


125 


of  bacilli.  In  twenty-four  hours  small  round  colonies  are  found 
which  seem  to  arrange  themselves  concentrically.  The  growth 
becomes  more  abundant,  and  the  individual  colonies  larger  and 
yellowish.  On  blood-coagidum  the  growth 
is  usually  gray  and  the  margins  of  the 
culture  crenated.  Often  a  diagnosis  can 
be  made  in  four  hours  if  the  serum-tubes 
are  kept  in  a  brood-oven. 

Serum-agar. — Joos  finds  senim-agar 
better  than  Loffler's  serum:  300  c.c. 
blood-serum  mixed  with  50  c.c.  normal 
soda  solution  and  150  c.c.  water,  heated 
in  water-bath  for  two  to  three  hours  at 
60°  to  70°  C.,  then  raised  to  100°  C.,  or 
in  steam-chest  one-half  hour.  Then  500 
c.c.  peptone  bouillon  (slightly  alkaline) 
and  20  gm.  agar.  When  the  agar  is 
dissolved  by  heat,  avoiding  prolonged 
boiling,  the  mixture  is  filtered  and  steril- 
ized one-quarter  hour  at  100°  to  110°  C. 
in  autoclave;  then  poured  into  Petri 
dishes.  Streptococci  do  not  grow  on 
this  medium,  whereas  diphtheria  bacilli 
will  grow  in  from  six  to  twelve  hours. 

Bouillon. — In  bouillon  an  abundant 
growth  takes  place,  and  this  medium  is 
used  to  obtain  the  toxins. 

Staining.  —  Is    positive    by    Gram's 
method.    Stained  best  with  Loffler's-alka- 
line  methylene-blue.     Neisser's   double 
stain    (see    p.  34)    shows    granules,   blue   black,    and   body, 
brown. 

Pathogenesis. — By  inoculation,  animals,  which  naturally  are 
not  subject  to  diphtheria,  have  had  diphtheritic  processes 
develop  at  the  site  of  infection;  hemorrhagic  edema  then 
follows,  and  death. 

No  agglutinins  are  developed  in  the  serum. 


Fig.  63. — Bacillus 
diphtherias;  agar-agar 
culture  (photograph  by 
Dr.  Henry  Koplik). 


126          ESSENTIALS  OF  BACTERIOLOGY 

In  rabbits  paralyses  develop,  and  when  the  inoculation  occurs 
upon  the  trachea,  all  the  prominent  symptoms  of  diphtheria 
show  themselves. 

Manner  of  Infection  in  Man. — The  exact  way  is  not  yet 
known.  It  is  supposed  that  the  mucous  membrane,  altered  in 
some  manner,  the  diphtheria  bacillus  then  gains  entrance  and 
the  disease  develops.  The  bacilli  may  be  found  in  healthy 
individuals  who  may  act  as  a  source  of  infection  to  susceptible 
individuals  without  themselves  becoming  infected. 

Prevalence  of  Bacillus  Diphtheria. — Examinations  made  on 
a  large  scale  of  the  throats  of  supposedly  healthy  individuals 


Fig.  64. — Bacillus  diphtheriae,  from  a  pure  culture. 

have  shown  that  the  Bacillus  diphtheriae  is  rather  widely  dis- 
tributed. Not  only  does  it  linger  for  many  weeks  in  the  throats 
of  persons  recently  recovered  from  the  disease,  but  it  is  found 
in  the  care-takers,  nurses,  etc.,  and  there  are  allied  organisms, 
with  more  or  less  pathogenicity,  that  have  been  found  in 
atrophic  rhinitis,  in  conjunctivitis,  and  in  the  throats  of 
unexposed  normal  individuals.  The  pseudobacillus  of  Hoff- 
man is  believed  by  many  investigators  to  be  but  a  weakened 
diphtheria  bacillus  that  has  lost  its  toxic  power. 

Methods  of  Diagnosis. — A  small  piece  of  exudate  or  some 
secretion  from  pharynx,  tonsil,  or  nares  is  obtained  on  a  sterile 
cotton  swab  and  transferred,  as  soon  as  possible,  to  the  surface 


PATHOGENIC   BACTERIA 


127 


of  two  or  more  blood-serum  tubes  (if  these  are  not  available, 
the  swab  should  be  placed  in  a  sterile  test-tube  or  bottle, 
and  sent  to  the  laboratory  at  once).  The  inoculated  tubes 
are  placed  in  the  incubator  at  37°  C.,  and  examined  in  twelve 
hours.  If  a  growth  is  visible,  a  slide  is  made  and  stained  with 
Loffler's  and  Neisser's  stain,  and  if  bacilli  are  present,  with 
characteristic  granules,  the  diagnosis  of  diphtheria  is  most 
probable.  If  there  are  no  clinical  signs,  the  growth  should  be 


Fig.  65. — Bacillus  diphtherias,  from  a  culture  upon  blood-serum    (x  1000) 
(Frankel  and  Pfeiifer). 

tested  for  toxicity  by  inoculating  a  guinea-pig;  it  should  be 
grown  in  alkaline  sugar  bouillon  and  tested  in  two  days  for 
acid.  The  xerosis  and  Hoffman's  bacilli  are  not  pathogenic 
for  guinea-pigs  and  do  not  produce  an  acid  reaction  in  sugar 
media. 

Products. — But  it  is  not  the  mere  presence  of  the  bacillus  that 
gives  rise  to  all  trouble;  certain  products  which  they  generate 
get  into  the  system  and  produce  the  severe  constitutional  symp- 
toms. 


I2&  ESSENTIALS    OF    BACTERIOLOGY 

Roux  and  Yersin,  in  1888,  discovered  that  the  injection  of  the 
filtered  culture  bouillon  (that  is,  freed  of  all  diphtheria  bacilli) 
gave  rise  to  the  same  palsies  as  when  the  bacilli  themselves  were 
introduced. 

Toxins  of  Diphtheria. — The  toxins  may  be  separated  from 
three-weeks-old  bouillon  cultures  by  nitration.  They  are  not 
albumins  and  are  very  complex.  Ehrlich  claims  two  principles: 
one  he  calls  toxone.  The  other,  toxin,  the  toxone  produces  para- 
lytic symptoms,  and  appears  to  be  less  affected  by  antitoxin. 
The  toxins  are  highly  poisonous — o.ooi  c.c.  may  be  sufficient 
to  kill  a  guinea-pig  in  less  than  twenty-four  hours.  The  sub- 
stance is  unstable,  losing  its  toxic  power  gradually.  Heating 
at  58°  C.  for  two  hours  is  destructive,  but  drying  renders  it 
more  stable. 

Antitoxin. — Behring  found  that  animals  rendered  immune 
had  a  principle  in  their  blood  that  was  antagonistic  to  the 
development  of  the  toxin. 

Immunity. — Brieger  and  Frankel,  by  injecting  10  to  20  c.c. 
of  a  three-weeks-old  culture  of  diphtheria  bacilli,  which  had 
been  heated  at  70°  C.  for  one  hour,  produced  an  immunity  in 
guinea-pigs  against  the  virulent  form. 

The  strength  commonly  employed  in  human  beings  is  3000 
units,  and  as  much  as  20,000  units  may  be  given  without  detri- 
ment in  severe  cases.  If  this  amount  is  injected  into  a  child 
suffering  from  diphtheria  in  the  earlier  stages  (second  to  third 
day),  the  disease  is  often  arrested.  The  membrane  begins 
to  disappear,  and  in  two  or  three  days  has  vanished.  The  con- 
stitutional symptoms  are  likewise  greatly  influenced  by  the 
injection.  For  prophylaxis  and  immunizing  well  persons 
1000  to  1500  units  are  employed. 

In  such  conditions  as  asthma  severe  and  fatal  results  have 
followed  the  use  of  the  serum. 

The  antitoxin  has  no  influence  on  the  bacteria  themselves; 
their  virulence  and  length  of  residence  in  the  body  are  not  less- 
ened. 

The  toxin  generated  by  the  germ  is  supposed  to  be  neutralized 
by  the  antitoxin  and  prevented  from  injuring  the  body  tissues. 


PATHOGENIC  BACTERIA  1 29 

Preparation  of  Toxin. — The  bacillus  is  grown  in  muscle- 
sugar-free  bouillon  with  an  alkaline  reaction.  Acids  prevent 
toxin  formation.  There  should  be  a  free  supply  of  oxygen, 
and,  therefore,  large  shallow  flasks  are  used.  The  maximum 
toxicity  is  developed  in  seven  to  ten  days.  The  strength  should 
be  -5-^-5-  c.c.,  fatal  for  5oo-gram  guinea-pig. 

Preparation  of  Antitoxic  Serum. — Horses  are  rendered 
immune  by  gradually  increased  doses  of  diphtheria  toxin,  the 
power  of  the  toxin  having  first  been  standardized  by  its  neu- 
tralization with  some  standard  antitoxin  in  powdered  form. 
The  toxin  is  at  first  injected  subcutaneously,  then  intravenously, 
and  after  several  months'  treatment  a  resistance  is  obtained 
that  will  withstand  300  to  500  times  the  original  lethal  dose. 
The  horse  is  then  bled,  and  from  five  to  nine  liters  withdrawn, 
this  is  then  allowed  to  coagulate,  and  under  very  careful 
precautions  the  serum  is  placed  in  sterile  packages,  its  strength 
having  first  been  compared  with  a  standard  furnished  by  the 
United  States  Government. 

Antitoxic  Unit. — A  normal  antitoxic  serum  is  one  that 
contains  in  each  cubic  centimeter  one  immunity  unit.  An 
immunity  unit,  according  to  Ehrlich,  is  the  amount  of  antitoxic 
serum  which  will  neutralize  100  times  the  nr'nimum  lethal 
dose  of  toxin,  when  serum  and  toxin  mixed  and  injected  into 
a  25o-gram  guinea-pig  does  not  cause  death  in  four  days.  Thus, 
if  the  serum  will  protect  in  doses  of  -^  c.c.,  then  each  cubic  cen- 
timeter has  50  units  power,  and  20  c.c.  will  contain  1000  units, 
or  will  be  sufficient  to  neutralize  an  amount  of  toxin  that  would 
be  fatal  for  25,000  kilos  (12,500  pounds)  of  guinea-pigs,  or 
100,000  pigs  weighing  250  grams  each. 

Streptococcus  in  Diphtheria. — Streptococci  have  been 
found  quite  constant  in  diphtheria,  but  they  resemble  the 
Streptococcus  pyogenes,  and  have  no  specific  action. 

Bacillus  of  Typhoid  or  Enteric  Fever  (Eberth-Gaffky)  .— 
Origin. — Eberth  found  this  bacillus  in  the  spleen  and  lymphatic 
glands  in  the  year  1880,  and  Gaffky  isolated  and  cultivated 
the  same  four  years  later. 

Form. — Rods  with  rounded  ends  about  three  times  as  long  as 


130  ESSENTIALS    OF    BACTERIOLOGY 

they  are  broad.  Usually  solitary  in  tissue-sections,  but  in  arti- 
ficial cultures  found  in  long  threads.  Flagella  on  the  side. 

Properties. — They  are  very  motile;  they  take  the  anilin  dyes 
less  deeply  than  some  similar  bacilli.  Spores  have  not  yet  been 
found;  they  do  not  liquefy  gelatin. 

Growth. — They  are  facultative  anaerobic;  grow  best  at  37° 
C.,  but  can  also  develop  at  ordinary  room  temperature.  All 
nutrient  media  can  be  used  as  culture-ground.  They  develop 
chiefly  on  the  surface,  and  very  slowly.  Repeated  freezing  and 


Fig.  66. — Bacillus  typhi,  from  an  agar-agar  culture  six  hours  old,  showing  the 
flagella  stained  by  Loffler's  method  (x  1000)  (Frankel  and  Pfeiffer). 

thawing  do  not  affect  the  vitality  of  the  germ,  and  phenol 
in  i  to  2  per  cent,  solution  has  no  effect  on  it.  A  ten-minute 
exposure  to  60°  C.  is  invariably  fatal. 

Colonies  on  Gelatin  Plates. — Two  forms;  the  ones  near  the 
surface  spread  out  like  a  leaf,  transparent,  with  bluish  fluor- 
escence. The  deeper  ones  resemble  whetstone  crystals  of  uric 
acid,  with  the  same  yellowish  tinge. 

In  five  days  they  attain  to  3  millimeters  in  diameter. 

On  Potato  Gelatin.— The  colonies  do  not  have  the  yellow 


PATHOGENIC   BACTERIA 


color,  they  are  transparent;  later  on  they  become  dark  brown 
with  green  iridescence. 

Stab-cultures. — Mainly  on  the  surface,  a  pearly  layer. 

Stroke  Cultures. — A  transparent  thick  layer. 

Potato. — The  growth  here  is  quite  characteristic.  At  37°  C. 
in  forty-eight  hours  a  moist,  transparent  film  is  formed  over 
the  whole  surface,  but  so  transparent  that  it  can  hardly  be  seen 
without  close  observation.  If  a  small  portion  of  this  is  placed 
under  a  microscope,  it  will  be  seen  swarming  with  bacilli. 

The  growth  never  becomes  more  prominent;  the  potato  must 
have  a  neutral  or  acid  reaction. 


Fig.  67. — Typhoid     fever     bacillus 
in  pure  culture  (x  650). 


Fig.  68. — Colonies  of  typhoid 
bacilli  three  days  old  (x  100) 
(Friinkel  and  Pfeiffer). 


Milk. — The  bacteria  grow  very  well  in  milk,  producing  a 
slightly  acid  reaction,  but  no  coagulation. 

Phenol  Gelatin. — Gelatin  which  has  added  to  it  -^  per  cent, 
phenol  will  allow  the  typhoid  bacillus  to  develop,  other  similar 
bacilli  being  destroyed. 

Glucose  Gelatin. — In  glucose  gelatin  there  is  no  gas-produc- 
tion. Indol  is  likewise  not  generated  by  the  typhoid  bacillus, 
whereas  it  is  by  the  colon  bacillus.  On  Eisner's  potato-gelatin 
the  colon  bacillus  and  the  typhoid  bacillus  grow  readily.  The 
medium  of  Hiss  is  of  great  assistance  in  isolating  the  germ. 

The  Gruber-Widal  blood-serum  test,  or,  as  it  is  otherwise 
known,  the  agglutination  phenomenon  (Fig.  69),  has  the 
following  history: 


132  ESSENTIALS    OF    BACTERIOLOGY 

About  1889,  Charrin  and  Roger  observed  in  the  serum  of 
immunized  animals  that  the  Bacillus  pyocyaneus  arranged 
itself  in  little  clumps.  Other  investigators  reported  the  same 
thing  for  other  bacteria,  and  Metchnikoff  added  that  motility 
was  destroyed. 

In  1895  Bordet  showed  that  the  serum  of  cholera-immunized 
animals,  when  mixed  with  bouillon  cultures  of  cholera  spirilla, 
affected  their  motility  and  caused  them  to  form  masses,  or 
"Klumpen,"  as  the  Germans  call  it. 


Fig.  69. — The  Widal  agglutination  reaction  (Slater  and  Spitta). 

R.  Pfeiffer,  in  the  same  year,  showed  that  the  introduction  of 
immune  serum  at  the  same  time  with  virulent  cholera  spirilla 
into  the  peritoneum  of  guinea-pigs,  prevented  infection  from 
taking  place,  and  the  spirilla  were  transformed  into  granular 
masses.  He  likewise  showed  this  reaction  to  be  specific,  the 
serum  of  cholera-immune  animals  acting  only  on  cholera 
vibrio,  and  hence  he  suggested  using  the  serum  as  a  means 
of  diagnosis  for  the  cholera  vibrio  and  typhoid  bacillus.  Gru- 
ber  about  the  same  time  made  some  studies  upon  the  use  of 
this  serum  property  in  differentiating  bacteria,  but  it  was  con- 
sidered as  yet  a  property  connected  in  some  way  with  immunity. 


PATHOGENIC   BACTERIA  133 

In  1896  Widal  and  Griinbaum,  working  separately,  devel- 
oped what  is  now  spoken  of  as  the  "Widal  serum  test,"  or 
"Widal  reaction."  It  consists  in  testing  a  drop  of  blood  of  a 
patient  suspected  of  having  typhoid  fever,  by  mixing  a  dilution 
of  it  with  a  drop  of  a  fresh  bouillon  culture  of  typhoid  bacilli, 
and  examining  the  mixture  in  a  hanging  drop  under  the  micro- 
scope. Within  fifteen  minutes  to  an  hour  the  motility  of  the 
bacilli  will  cease,  and  they  will  have  arranged  themselves  into 
clusters,  as  if  stuck  or  glued  together.  If  this  reaction  occurs 
within  an  hour,  and  with  the  proper  dilution  of  the  serum,  the 
case  is  one  of  typhoid.  Widal  first  used  the  serum  of  the  blood; 
this  has  been  modified  so  that  even  a  drop  of  dried  blood  is  suf- 
ficient. The  method  as  applied  in  city  laboratories  is  as  follows : 
The  physician  is  told  to  clean  the  finger  of  the  patient  with 
water  (no  germicides),  and  with  a  needle  draw  a  drop  of  blood 
on  to  a  piece  of  ordinary  note-paper.  This  is  then  sent  to  the 
laboratory;  the  paper  with  the  dried  blood  is  soaked  for  a  few 
minutes  in  a  watch-glass  containing  4  drops  of  clean  water,  thus 
obtaining  a  dilution  of  i  15.  One  drop  of  this  is  then  mixed 
with  one  drop  of  a  bouillon  culture  of  typhoid  bacilli  of  about 
twenty-four-hours'  growth,  and  examined  under  the  microscope 
in  the  hanging  drop.  Weaker  dilutions  of  the  serum  have  been 
recommended  (i  150),  and  this  should  be  used  in  cases  of 
doubt.  So  far,  about  95  per  cent,  of  the  cases  examined,  and 
which  clinically  were  considered  typhoid  fever,  have  given  a 
positive  reaction.  It  is  not  often  present  until  the  fifth  day  of 
the  fever,  and  disappears  usually  within  a  year,  though  in  some 
individuals  it  has  been  found  ten  years  after  an  attack  of  the 
disease. 

The  agglutinating  properties  have  been  found  in  nearly  all 
the  secretions  of  the  body — tears,  urine,  milk,  pleuritic  effusions, 
serous  fluid  from  blisters,  etc. 

There  is  no  relation  between  the  reaction  and  the  bactericidal 
power  of  the  serum;  the  agglutination  is  not  a  destruction.  The 
agglutinating  power  is  active,  though  the  blood  be  dried  and 
sealed  up  for  months.  It  seems  to  have  no  direct  relation  with 
the  question  of  immunity,  since  it  occurs  at  the  height  of  the 


134  ESSENTIALS    OF    BACTERIOLOGY 

disease,  and  intense  agglutinating  serum  may  be  had  in  severe 
cases  and  in  cases  with  relapses.  A  negative  result  does  not 
exclude  typhoid. 

The  test  is  quantitative — i.  e.,  it  depends  upon  the  dilution  of 
the  blood-serum,  since  the  serum  of  healthy  persons  in  strong 
dilution  will  cause  agglutination  and  loss  of  motility. 

The  test  must  occur  within  a  certain  limit  of  time  to  be  of 
value,  since  agglutination  is  liable  to  appear  of  itself  with  non- 
typhoid  serums  after  a  period  of  an  hour. 

A  serum  in  a  dilution  of  i  :  30  causing  complete  clumping 
in  half  an  hour  is  most  likely  typhoid. 

The  culture  must  be  kept  in  a  vigorous  condition  by  frequent 
subplanting,  and  must  be  tested  occasionally  with  normal 
serum.  Cultures  kept  in  an  incubator  for  a  long  time  tend  to 
adhere  in  clumps  naturally. 

Sedimentation  Test. — In  a  test-tube  of  bacterial  emulsion 
and  diluted  serum,  if  the  reaction  is  positive,  there  will  be  a 
flocculent  precipitate  or  sediment  at  the  bottom  of  the  tube, 
the  upper  part  remaining  clear.  Compared  with  normal 
serum,  the  cloudiness  is  diffuse. 

As  a  clinical  test  of  the  disease  it  has  considerable  value, 
although  operative  at  a  time  when  other  symptoms  have  devel- 
oped sufficiently  to  determine  the  diagnosis. 

Staining. — Colored  with  the  ordinary  anilin  dyes,  when  they, 
are  warmed;  since  they  are  easily  decolorized,  acids  should  be 
avoided. 

Gram's  method  is  not  applicable.  Tissue  sections  stained  as 
follows: 

Alkaline  methylene-blue i  hour 

Alcohol 5  seconds 

Anilin-oil 5  minutes 

Turpentine-oil i  minute 

Xylol  and  Canada  balsam 

Such  a  specimen  should  first  be  examined  with  low  power,  to 
focus  little  colored  masses,  then  examined  with  immersion  lens; 
these  masses  will  be  found  composed  of  bacilli. 


PATHOGENIC   BACTERIA  135 

Similar  Bacteria. — The  N eapolitanus  bacillus  of  Emmerich, 
or  feces  bacillus  of  Brieger,  resembles  the  typhoid  bacillus  in 
many  ways,  the  colonies  being  the  same  and  its  structure 
similar.  But  the  growth  on  potato  is  very  different;  a  thick, 
yellow,  pasty  layer  is  formed  thereon. 

The  colon  bacillus  not  only  resembles  the  typhoid  germ  in 
form,  but  also  in  some  of  the  pathologic  processes  produced. 
For  points  of  resemblance  and  difference  see  Bacillus  coli  com- 
munis. 

Typhoid  Bacilli  in  Water. — Although  all  evidence  shows  that 
the  water-supply  is  a  frequent  source  of  infection,  very  few 
persons  have  ever  isolated  the  typhoid  bacillus  from  such  an 
infected  source.  The  earlier  reports  show  that  no  account 
was  taken  of  Bacillus  coli,  which  is  usually  present  in  polluted 
waters.  (See  Water  Analysis.) 

Pathogenesis. — Lower  animals  do  not  have  enteric  fever, 
though  their  death  has  been  caused  by  injection  of  the  bacilli 
into  the  veins  of  the  ear. 

In  man  the  bacillus  has  been  found  in  the  urine,  blood,  spu- 
tum, milk,  intestinal  discharges,  roseolar  spots,  and  in  various 
organs,  as  spleen,  liver,  lymphatic  glands,  and  intestinal  villi. 

It  is  found  in  secretions  several  days  after  the  attack  has  sub- 
sided. It  is  found  only  in  this  disease,  and  regularly. 

Typhoid  Carriers. — Some  individuals  retain  a  culture  of 
the  bacilli  in  the  gall-bladder  for  years,  and  manufacture,  or 
at  least  expel,  true  virulent  bacilli  through  the  feces  continually. 
Such  persons  have  infected  other  individuals  without  suffering 
any  inconvenience  themselves.  Some  forms  of  chronic  inflam- 
mation, as  cholecystitis  and  appendicitis,  have  been  caused  by 
the  typhoid  bacillus. 

Way  of  Infection. — The  bacilli  in  the  dejecta  of  the  diseased 
person  find  their  way  into  drinking-water,  milk,  or  dirty  clothes, 
and  so  into  the  alimentary  tract  of  a  person  predisposed  to  the 
disease.  Flies  act  as  conveyors  by  infecting  food.  The  bacilli 
enter  the  blood  through  the  lymphatics,  and  so  become  lodged 
in  various  organs.  They  are  quite  resistant,  living  for  some 
time  in  the  soil  and  water,  and  are  not  affected,  as  other  organ- 


136  ESSENTIALS    OF    BACTERIOLOGY 

isms,  by  phenol.  An  epidemic  has  been  traced  to  the  eating 
of  oysters  taken  from  contaminated  water. 

Persistence  in  Water. — Franckland  kept  bacilli  alive  in  water, 
sterilized  by  heat,  seventy-five  days;  in  filtered  water  at  19°  C., 
five  days;  at  6°  C.,  twelve  days.  In  ordinary  water  they  are 
likely  to  be  destroyed  in  a  few  days  by  the  overgrowth  of  other 
bacteria. 

Products. — Brieger  found  a  ptomain  in  the  cultures,  which  he 
named  typhotoxin,  with  the  formula  C9H17NO2.  It  has  no 
specific  action.  A  toxalbumin  insoluble  in  water  has  also  been 
isolated,  but,  as  experiment  animals  are  immune  to  the  disease, 
no  definite  actions  have  yet  been  determined. 

The  cultures,  when  old,  show  an  acid  reaction. 

Vaccines. — Dead  cultures  of  tested  virulence  are  injected 
subcutaneously.  This  causes  the  blood  to  show  agglutination 
reaction,  increased  opsonic  index,  and  a  heightened  immunity 
to  the  disease.  In  several  thousand  inoculations  observed 
by  Wright  it  is  claimed  the  disease  was  much  lessened  in 
severity  and  the  tendency  to  infection  much  reduced. 

Typhoid  Bacilli  in  Blood. — Conradi,  Busquet,  Coleman, 
and  Buxton,  and  others  have  found  the  bacilli  in  the  blood  of 
every  patient  by  the  following  method.  A  mixture  of  ox-bile, 
90  c.c.,  glycerin,  10  c.c.,  and  peptone,  2  gm.,  is  distributed  into 
20  c.c.  flasks  and  sterilized.  Ten  cubic  centimeters  of  blood  is 
drawn  from  the  elbow  into  a  glass  syringe  and  divided  among 
three  flasks.  These  are  incubated,  and  in  twenty-four  hours 
litmus-lactose  agar  plates  are  inoculated  on  the  surface  by  a 
stroke  from  the  flasks.  A  growth  is  obtained  in  five  or  six 
hours. 

If  the  growth  is  a  bacillus  which  has  not  reddened  the 
medium,  it  is  tested  for  the  Widal  reaction  with  immune 
serum.  The  diagnosis  has  been  made  as  early  as  the  second 
day. 

Paracolon  or  paratyphoid  bacilli  are  members  of  the  colon 
group  recently  described  by  Widal,  Gwyn,  Schottmiiller,  and 
others.  They  are  of  importance,  since  they  produce  fevers 
clinically  resembling  a  mild  form  of  typhoid,  but  which  are 


PATHOGENIC   BACTERIA  137 

rarely  fatal.  They  may  be  the  sole  cause  of  the  disease, 
and  also  occur  together  with  the  typhoid  bacillus  in  mixed 
and  secondary  infections.  Morphologically,  they  resemble  the 
typhoid  bacillus,  but  differ  from  it  culturally  and  give  their 
own  serum  reactions  with  the  blood  of  affected  patients.  They 
ferment  glucose,  but  not  lactose  or  saccharose;  litmus  milk  at 
first  becomes  acid,  but  later  grows  alkaline  and  is  not  coagu- 
lated. On  potato  a  slight  visible  growth  occurs;  indol  is  usually 
not  formed.  Typhoid  serums  do  not  agglutinate  paracolon 
bacilli,  and  vice  versa;  also  different  paracolon  infections  may 
not  agglutinate  each  other.  The  Bacillus  enteritidis  of 
Gartner  is  a  related  form. 

Bacillus  psittacosis  is  an  allied  form  occurring  in  parrots, 
and  producing  hemorrhagic  septicemia  in  them  and  other  experi- 
ment animals.  The  disease  is  readily  communicated  to  man 
from  the  affected  birds,  and  causes,  after  ten  days'  incubation, 
a  disease,  the  chief  symptoms  of  which  are  fever,  delirium, 
vomiting,  diarrhea,  and  albuminuria,  about  a  third  of  the 
cases  ending  fatally.  The  organism  is  agglutinated  by  strong 
dilutions  of  typhoid  serum,  but  the  clumping  is  incomplete  and 
the  bacillus  differs  further  from  the  typhoid  bacillus  in  its 
growth  on  potato  and  in  the  nature  of  the  infection  produced. 

Bacillus  Coli  Communis  (Escherich). — Found  in  human 
feces,  intestinal  canal  of  most  animals,  in  pus  and  water. 

Form. — Short  rods  with  very  slow  movement,  often  asso- 
ciated in  little  masses  resembling  the  typhoid  germ,  flagellated, 
not  forming  spores  (Fig.  70) . 

Properties. — Does  not  liquefy  gelatin,  causes  fermentation  in 
saccharine  solutions  in  the  absence  of  oxygen,  produces  acid 
fermentation  in  milk,  causes  formation  of  indol  in  peptone 
solutions;  its  optimum  temperature  for  growth  is  37°  C. 

Growth. — On  potato  a  thick,  moist,  yellow-colored  growth; 
on  agar  a  gray-white  growth;  on  gelatin  a  growth  similar  to 
typhoid.  It  can  also  develop  on  phenol-gelatin,  and  withstands 
a  temperature  of  45°  C.  without  its  growth  being  destroyed. 

Patho  gene  sis. — Inoculated  into  rabbits  or  guinea-pigs,  death 
follows  in  from  one  to  three  days,  the  symptoms  being  those  of 


138 


ESSENTIALS    OF    BACTERIOLOGY 


diarrhea  and  coma;  after  death  tumefactions  of  Peyer's  patches 
and  other  parts  of  the  intestine;  perforations  into  peritoneal 
cavity,  the  blood  containing  a  large  number  of  bacilli. 

With  the  blood  of  immunized  animals  a  serum  reaction 
similar  to  that  of  typhoid  fever  may  be  obtained  with  cultures 
of  colon  bacilli.  The  colon  bacillus  is  held  responsible  for  most 
of  the  complications  of  typhoid  fever,  such  as  peritonitis, 
cholangitis,  etc.,  by  many  writers. 


Fig.  70. — Bacillus    coli    communis,    from    an    agar-agar    culture    (x  1000) 
(Itzerott  and  Niemann). 

Epidemics  of  a  cholera  or  dysentery  nature,  called  by  Esche- 
rich  colitis  contagiosa,  and  due  to  infection  of  water  and  food, 
have  been  noted  by  a  number  of  writers.  The  onset  is  very 
sudden  and  prostrating,  though  not  fatal. 

Many  other  forms  of  suppuration  are  associated  with  the 
presence  of  Bacillus  coli. 

Staining. — Ordinary  stains;  does  not  take  Gram. 

Site. — The  bacillus  has  been  found  very  constant  in  acute 
peritonitis  and  in  cholera  nostras.  Its  presence  in  water  would 
indicate  fecal  contamination,  as  it  is  normally  present  in  the 
intestine. 


PATHOGENIC    BACTERIA  139 

Points  of  Resemblance  between  Bacillus  Typhi  and  Bacillus 
Coli  Communis. — One,  microscopic  appearance;  two,  agar  and 
gelatin  cultures;  three,  sometimes  growth  on  potato  the  same; 
four,  staining  peculiarities;  five,  resistance  to  phenol. 

Points  of  Difference: 

Colon  Bacillus.  Typhoid  Bacillus. 

Less  motile.  Actively  motile. 

Gelatin  colonies  develop  more  Develop  more  slowly. 

rapidly. 

Produces  gas  on  dextrose  or  Does  not. 

lactose  media. 

Coagulates  milk.  Does  not. 

Produces  indol.  Does  not. 

Growth  on  potato  visible.  Invisible. 

Changes  neutral  red  to  yellow.  Does  not  reduce  neutral  red. 

Differences  are  also  noted  in  the  growth  on  special  media, 
such  as  those  of  Hiss  and  Eisner. 

Varieties. — By  some  bacteriologists  the  following  bacilli  are 
all  considered  forms  of  the  colon  bacillus.  Bacillus  lactis 
aerogenes  of  Escherich;  B.  cavicida,  of  Brieger;  B.  neapoli- 
tanus  of  Emmerich;  B.  enteritidis  of  Gartner,  and,  together 
with  some  other  allied  organisms,  they  are  spoken  of  as  the 
" colon  group." 

Saccharolytic  Bacteria. — These  are  organisms  found  in  water 
that  produce  fermentation  in  sugar  broth  and  form  acids,  but 
do  not  give  the  reaction  for  indol.  Rivas  has  perfected  some 
chemical  tests  that  he  claims  distinguish  this  group  from  true 
Bacillus  coli,  but  whether  they  possess  any  different  pathogenic 
properties  has  not  been  determined. 

Bacillus  Botulinus  (Van  Ermengem) . — An  anaerobic  ba- 
cillus cultivated  by  Van  Ermengem  in  1896  from  ham  which 
had  caused  poisoning. 

Form. — A  large  bacillus  with  rounded  or  spindle-shaped 
ends,  and  often  with  oval  terminal  spores,  motile,  with  lateral 
flagella. 


I4O  ESSENTIALS    OF    BACTERIOLOGY 

Staining. — Gram  positive,  easily  stained  with  ordinary  dyes. 

Growth. — Strictly  anaerobic.  Forms  abundant  gas  in  glu- 
cose, gelatin,  and  liquefies  cultures,  producing  butyric  acid 
odor.  Best  temperature  between  20°  and  30°  C. 

Pathogenesis. — Produces  a  powerful  toxin  in  the  tissues, 
like  the  tetanus  bacillus.  This  toxin  may  be  present  in  the 
affected  meat  without  causing  decomposition,  and  thus  give 
rise  to  poisoning. 

Bacillus  enteritidis  of  Gartne'r  has  likewise  been  considered 
a  cause  of  meat  and  sausage  poisoning. 


CHAPTER  XIX 

PATHOGENIC  BACTERIA   (Continued) 

Spirillum  Choleras  (Koch)  (Comma  Bacillus  of  Cholera) . 

— Origin. — Koch,   as  a  member  of  the  German  expedition 
sent  to  India,  in  1883,  to  study  cholera,  found  this  micro-organ- 
ism in  the  intestinal  contents  of  cholera 
patients,  and  by  further  experiments 
identified  it  with  the  disease. 

Form. — The  spirillum  as  seen  ordi- 
narily appears  as  a  short,  arc-like  body, 
about  half  the  size  of  a  tubercle  bacillus, 
but  when  seen  in  large  groups,  spirals 
are  formed,  each  little  arc  appearing 
Fig.  71.— Comma  ba-  then  as  but  a  segment,  a  vibrio;  each 

cillus,  pure  culture  (x  600).  .  ,.  ,  ., 

arc  is  about  three  times  as  long  as  it 

is  broad,  and  possesses  a  flagellum  at  one  or  more,  rarely 
both,  ends. 

Properties. — The  spirilla  are  very  motile;  liquefy  gelatin. 
They  are  easily  affected  by  heat  and  dryness.  Spores  have  not 
been  found,  though  some  (Hiippe)  claim  arthrospores,  but  these 
bodies  represent  only  degenerative  changes. 

Growth. — At  ordinary  temperatures  on  all  nutrient  media 


PATHOGENIC    BACTERIA 


that  have  an  alkaline  or  neutral  reaction.     They  are   facul- 
tative anaerobic. 

Colonies,  Gelatin. — After  twenty-four  hours,  small  white 
points  which  gradually  come  to  the  surface,  the  gelatin  being 
slowly  liquefied,  a  funnel-shaped  cavity  formed,  holding  the 
colony  in  its  narrow  part,  at  the  bottom,  and  on  the  fifth  day 
all  the  gelatin  is  liquid.  If  the  colonies  of  three  days'  growth 
are  placed  under  microscope,  they  appear  as  if  composed  of 
small  bits  of  frosted  glass  with  sharp  irregular  points. 

Stab-culture. — After  thirty  hours  a  growth  can  be  distin- 
guished along  the  needle-track,  and  on  the  surface  a  little 
cavity  is  formed,  filled  by  a  bubble  of  air,  and  this  liquefaction 
proceeds  until,  on  the  sixth  day,  it  has  reached  the  sides  of 
the  tube,  tapering,  funnel-shaped,  to  the  bottom  of  the  tube. 
After  several  weeks  the 
spirilla  are  found  in  little 
collections  at  the  bottom 
of  the  fluid  gelatin.  In 
eight  weeks  the  bacilli 
have  perished. 

Agar. — Stroke  cultures. 
A  shiny  white  layer  which 
lasts  many  months. 

Potato. — A  yellow, 
honey-like,  transparent 
layer,  if  the  potato  is 
kept  at  animal  heat. 

Bouillon. — A  wrinkled 
scum  is  soon  formed  in 
bouillon.  The  spirilla  live 
well  and  grow  in  sterilized 
milk  and  sterilized  water, 


Fig.  72. — Cholera  colonies  after  thirty  hours 
(X  100)  (Frankel  and  Pfeiffer). 


remaining  virulent  in  the  latter  for  many  months.  In  ordi- 
nary water  the  bacteria  present  are  destructive  to  the  comma 
bacilli,  and  they  die  in  a  few  days. 

Dunham's  Peptone  Solution. — Useful  for  the  development  of 
nitrites  and  the  indol  reaction. 


142  ESSENTIALS    OF    BACTERIOLOGY 

Widal's  serum  test,  as  used  in  typhoid,  is  applicable  in  the 
diagnosis  of  cholera,  using  cholera  cultures  in  place  of  the 
typhoid. 

Staining. — They  are  colored  well  with  watery  anilin  solu- 
tions. The  flagella  can  be  well  seen  by  staining  according  to 
the  flagella  stain. 

Patho genesis. — Experiment  animals  are  not  subject  to  cholera 
Asiatica,  but  by  overcoming  two  obstacles,  Koch  has  produced 
choleraic  symptoms  in  guinea-pigs.  Nicati  and  Rietsch  pre- 
vented peristalsis  and  avoided  the  acidity  of  the  stomach-juices 
by  direct  injection  into  the  duodenum,  after  tying  the  gall-duct. 
Koch  alkalinizes  the  gastric  juice  with  5  c.c.  of  5  per  cent, 
solution  of  sodium  carbonate,  and  then  injecting  2  grams  of 
opium  tincture  for  every  300  grams  of  weight  into  the  peritoneal 
cavity,  paralyzes  peristalsis.  The  cholera  culture  then  intro- 
duced through  a  stomach-tube,  the  animals  die  in  forty-eight 
hours,  presenting  the  same  symptoms  in  the  appearance  of  the 
intestines  as  in  cholera  patients,  the  serous  effusion  containing 
great  numbers  of  spirilla. 

Manner  of  Infection  in  Man. — Usually  through  the  alimen- . 
tary  tract,  with  the  food  or  drink,  the  intestinal  discharges  of 
cholera  patients  having  found  entrance  into  the  source  of  drink- 
ing water.  Soiled  clothes  to  fingers,  fingers  to  the  mouth,  etc.; 
torpid  catarrhal  affection  of  the  digestive  tract  predisposing. 
The  spirilla  are  not  found  in  the  blood  or  any  organ  other  than 
the  intestines,  the  tissue  of  the  small  intestines.  They  are  also 
found  in  the  vomit  and  the  intestinal  contents. 

Products. — "Cholera  red."  When  chemically  pure  nitric  or 
sulphuric  acid  is  added  to  nutrient  peptone  cultures  of  the 
cholera  bacillus,  a  rose-red  color  is  produced.  This  will  not  take 
place  with  other  bacilli  unless  nitrous  acid  is  present.  The 
cholera  bacillus  forms  nitrites  from  the  nitrates  present  in  the 
media,  and  also  indol.  The  mineral  acid  splits  the  nitrites, 
setting  free  nitrous  acid,  which,  with  the  indol,  forms  the  red 
reaction.  This  pigment  has  been  isolated  and  extracted  and 
called  " cholera  red."  A  ptomain,  identical  with  cadaverin,  and 
several  other  alkaloids,  have  been  obtained  from  the  cultures. 


PATHOGENIC   BACTERIA  143 

A  toxalbumin  and  a  toxic  peptone  have  been  isolated,  but  no 
special  actions  ascribed  to  them. 

Detection  of  Cholera  Organisms  in  Drinking-water. — When 
a  few  bacteria  are  supposed  to  be  present  in  fecal  matter  or 
drinking-water,  it  is  best  to  add  a  large  quantity  of  the  material 
(200  c.c.  of  drinking-water)  to  about  10  c.c.  of  bouillon  or 
peptone-water,  and  place  the  mixture  for  twenty-four  hours  in 
an  incubator,  which  will  cause  rapid  reproduction,  and  then 
the  organisms  can  be  readily  discovered. 


Fig-  73. — Comma  bacillus  in  mucus,  from  a  case  of  Asiatic  cholera. 

Protective  Vaccines. — Virulent  cultures  killed  by  heat  have 
shown  protective  power  and  were  used  extensively  during  an 
epidemic  in  Japan. 

Haffkine  has  obtained  a  great  reduction  in  mortality  in 
cholera  regions  by  the  use  of  anticholera  vaccines  as  protective 
and  curative  measures. 

Cholera  Immunity  of  Pfeiffer. — Intraperitoneal,  subcuta- 
neous, and  intravenous  injections  of  living  or»  dead  cholera 
bacteria  cause  a  disease  in  animals  similar  to  the  cold  stage  of 
cholera.  Death  is  the  result  of  toxemia.  If  the  animal  lives, 


144 


ESSENTIALS    OF    BACTERIOLOGY 


PATHOGENIC   BACTERIA 


145 


146 


ESSENTIALS    OF    BACTERIOLOGY 


the  blood  has  protective  properties  of  a  specific  nature;  it  has 
bactericidal  properties  against  cholera  vibrio,  and  by  the 
injection  of  this  serum  into  non-immune  animals  it  renders 
them  immune.  The  blood-serum  of  convalescents  and  cholera- 
vaccinated  individuals  contains  the  same  bactericidal  sub- 
stances. 

Serum  therapy  has  not  been  success- 
ful. 

Bacteria  Similar  to  the  Spirillum  of 
Cholera.  —  Finkler-Prior  Vibrio,  or 
Spirillum  Finkleri. — Origin. — Found  in 
the  intestinal  contents  of  a  patient  suffer- 
ing from  cholera  Asiatica  in  1884,  by 
Finkler  and  Prior,  who  thought  it  iden- 
tical with  the  spirillum  of  cholera;  it 
differs  from  it,  however,  in  many  ways, 
and  has  been  found  in  healthy  persons. 


»..**rj^  v*>— v^> 


^  "~ 

"^5xV>y^ 


Fig.  84.— Spirillum  Finkleri  (x  700)  (Fliigge). 


Fig.  85.— Stab  culture 
(Finkler-Prior). 


Form. — Somewhat  thicker  than  the  cholera  vibrio,  but  forms 
the  long  spirilla  less  often.  Has  flagella. 

Properties. — It  is  very  motile.  Liquefies  gelatin  in  a  short 
time. 

Growth. — It  grows  quickly  at  ordinary  room  temperature.  It 
is  facultative  aerobic. 

Colonies  on  Gelatin  Plates. — Round,  finely  granular  colonies, 
which  in  twenty-four  hours  are  ten  times  as  large  as  the  cholera 
colonies,  and  in  forty-eight  hours  the  whole  plate  is  liquefied, 
it  being  then  impossible  to  distinguish  any  separate  colonies. 


PATHOGENIC   BACTERIA  147 

The  microscopic  appearances  in  no  way  resemble  the  cholera 
colony. 

Stab-cultures. — The  gelatin  is  liquefied  from  above  down- 
ward, like  a  stocking  in  appearance,  and  in  three  days  is 
completely  liquid. 

Potato.— At  ordinary  temperature  a  thick  gray  layer  cover- 
ing the  whole  surface. 

Water. — It  soon  perishes  in  water. 

Staining. — Ordinary  anilin  dyes. 

Pathogenesis. — For  man  it  has  no  specific  action.  If  it  is 
injected  into  guinea-pigs,  prepared  as  described  under  the 
cholera  bacillus,  they  die,  the  intestines  having  a  foul  odor, 
and  the  bacilli  then  found  in  great  numbers. 

Spirillum  Tyrogenum  (Deneke). — Origin. — In  1885  Den- 
eke  found  in  old  cheese  a  spirillum  very  similar  in  appearance 
to  the  cholera  spirillum. 

Form. — The  same  as  the  cholera  vibrio. 

Properties. — Very  motile,  liquefy  gelatin. 

Growth. — They  grow  quicker  than  the  cholera,  and  slower 
than  the  Finkler;  they  are  also  facultative  aerobic. 

Colonies. — At  first  resemble  cholera  colonies;  have,  however, 
a  yellow-green  iridescence  and  are  more  irregular;  also  grow 
more  rapidly. 

Stab-cultures. — A  thick  line  along  the  needle-track  and  yellow 
colonies  forming  at  the  bottom;  on  the  surface  a  bubble  of  air 
similar  to  the  cholera.  The  gelatin  is  liquid  in  two  weeks;  on 
agar,  rapid  slimy  yellowish  growth. 

Potato. — At  brood-heat  a  thin  yellow  membrane,  but  not 
always  constant.  Staining,  as  cholera  bacillus. 

Pathogenesis. — When  injected  into  animals  prepared  as  for 
the  cholera  bacillus,  a  certain  number  die. 

Vibrio  Metchnikovi  (Gamaleia). — Origin. — In  the  intes- 
tines of  fowls  suffering  from  a  gastro-enteritis,  common  in 
Russia.  Gamaleia  found  a  spirillum  which  bears  so  close  a 
resemblance  to  the  cholera  bacillus,  both  in  form  and  growth, 
that  it  cannot  be  distinguished  by  these  characteristics  alone. 

Form. — As  cholera  bacillus. 


148         ESSENTIALS  OF  BACTERIOLOGY 

Growth. — Two  kinds  are  found  on  the  gelatin  plate — one  that 
is  identical  in  appearance  with  the  cholera  colony,  the  other 
more  liquefying,  resembling  the  Finkler  spirillum.  If  now  a 
second  plate  be  inoculated  from  either  one  of  these  forms,  both 
kinds  again  are  found  grown,  so  that  it  is  not  a  mixture  of  two 
bacilli;  on  agar,  brownish-yellow  growth. 

Stab-culture. — Similar  to  the  cholera  growth,  a  trifle  faster  in 
growing.  Staining,  as  cholera. 

Pathogenesis. — To  differentiate  it  from  cholera,  these  bacilli, 
when  injected  into  animals,  prove  very  fatal,  and  no  especial 
precautions  need  be  taken  to  make  the  animal  susceptible.  In 
the  pigeon,  guinea-pig,  and  chicken  a  hemorrhagic  edema  and 
a  septicemia  is  produced  which  has  been  called  "Vibrion 
septicemia."  The  blood  and  organs  contain  the  spirilla  in 
great  numbers. 

Products. — The  nitrites  are  formed  in  cultures  just  as  with 
cholera  bacillus,  and  the  red  reaction  produced  when  mineral 
acids  are  added  to  gelatin  cultures.  Certain  products  are  formed 
which,  when  injected,  give  immunity.  The  cultures  are  first 
heated  for  one-half  hour  at  100°  C.,  which  destroys  the 
germs,  and  then  this  sterilized  product  injected  (5  c.c.  of  a 
five-days'-old  sterilized  culture).  In  two  weeks  i  to  2  c.c.  of 
the  infected  blood  can  be  injected  without  causing  any  fatal 
result. 

Many  other  spirilla  resembling  the  spirillum  of  cholera  have 
been  isolated  from  drinking-waters  in  the  past  few  years, 
and  some  bacteriologists  are  inclined  to  consider  them  as 
varieties  of  the  true  cholera  spirillum,  which  require  only 
certain  conditions  to  make  them  pathogenic.  Among  these, 
besides  those  already  described,  are  Spirillum  berolinense,  S. 
dunbarii,  S.  danubicum,  S.  of  wernicke,  S.  bonhoffii,  S. 
weibeli,  S.  schuylkilliensis,  S.  milleri,  S.  aquatilis.  The  last 
two  are  non-pathogenic  for  experiment  animals. 

Bacteria  of  Pneumonia. — Two  forms  of  bacteria  have 
been  found  in  this  disease,  and  thought  at  different  times  to  be 
the  cause  of  the  same. 

Neither  one  of  them  is  constant  in  pneumonia;  and  since 


PATHOGENIC   BACTERIA  149 

many  other  pathologic  processes  have  shown  them,  they  can 
hardly  be  set  down  as  the  sole  cause  of  pneumonia. 

Klebs  in  1875  called  attention  to  the  presence  of  bacteria  in 
pneumonia,  and  in  1882  Friedlander  developed  a  bacillus  from 
the  lung  tissue  of  a  pneumonic  person,  which  he  thought  was  a 
coccus,  and  called  it  pneumococcus. 

In  1886  A.  Frankel  and  Weichselbaum  proved  that  this 
organism  was  not  constant — in  fact,  was  rare. 


Fig.  86. — Bacillus  pneumonias  of  Friedlander,  from  the  expectoration  of    a 
pneumonia  patient  (x  1000)  (Frankel  and  Pfeiffer). 

A.  Frankel  obtained  in  the  majority  of  cases  of  pneumonia 
an  organism  that  he  had  described  in  1884  under  the  name 
of  sputum-septicemia  micrococcus. 

Weichselbaum  called  it  Diplococcus  pneumonia,  and  believed 
it  to  be  the  real  cause  of  pneumonia.  It  has  been  found  in 
many  other  serous  inflammations,  and  also  in  the  mouths  of 
healthy  persons.  It  is  the  generally  accepted  organism  of  the 
disease,  and  can  be  isolated  from  nearly  all  cases  of  acute 
croupous  pneumonia.  It  is  found  in  about  three-quarters  of 
all  cases  of  pneumonia. 


ESSENTIALS    OF    BACTERIOLOGY 


Streptococcus  pyogenes  and  Staphylococcus  pyogenes  aureus 
have  been  found  in  some  cases. 

Pneumobacillus  (Pneumococcus)  (Friedlander)  .—Origin. 
— In  the  lung  of  a  croupous  pneumonia  person,  by  Friedlander, 
in  1882. 

Form. — Small,  almost  oval-shaped  rods,  nearly  as  wide  as 
they  are  long;  often  in  pairs,  they  were  at  first  believed  to  be 
cocci.     In  bouillon  cultures  the  rod  form  becomes  more  visible. 
In  tissues  each  bacillus  is  surrounded  by 
a  faint  capsule;  but  not  around  those  de- 
veloped in  artificial  cultures.     Spores  have 
not  been  found. 

Properties. — They  are  immobile;  do  not 
liquefy  gelatin.  A  gas  is  produced  in 
gelatin  cultures.  Ferment  sugar  solu- 
tions. 

Growth. — Grows  rapidly  on  all  media  at 
ordinary  temperature;  is  facultative  aerobic. 
Colonies. — On  gelatin  plates.  Small  white 
round  colonies,  reaching  the  surface  in  the 
course  of  three  or  four  days;  appearing 
then  as  little  buttons,  with  a  porcelain- 
like  shimmer,  the  edges  smooth. 

Stab-culture. — A  growth  along  the  needle- 
track,  but  on  the  surface  a  button-like  pro- 
jection, which  gives  to  the  growth  the  ap- 
pearance of  a  nail  driven  into  the  gelatin, 
of  pnfumo7n^Bastab-  its  head  resting  on  the  surface;  therefore 
culture  (nail  culture),  such  cultures  are  called  "nail  cultures." 
(See  Fig.  87.)  Old  cultures  are  colored 
brown  and  contain  bubbles  of  gas;  on  agar  and  blood-serum, 
a  thick  white  viscid  growth. 

Potato. — A  yellow,  moist  layer  in  a  few  days  at  brood-heat. 
Gas-bubbles  develop. 

Staining. — The  ordinary  anilin  stains.  The  sections  do  not 
take  Gram's  method:  are,  therefore,  not  suited  for  double 
staining. 


PATHOGENIC   BACTERIA  151 

Capsule. — Stained  as  follows: 
Cover-glasses: 

1.  Acetic  acid,  two  minutes. 

2.  Allow  acetic  acid  to  dry  by  blowing  air  upon  it  through 
a  glass  tube. 

3.  Saturated  anilin-water.     Gentian-violet,  ten  seconds. 

4.  Rinse  in  water.     Mount  in  Canada  balsam. 
For  sections: 

1.  Stain  for  twenty-four  hours  in  the  following,  warmed: 

Concentrated  alcoholic  gentian-violet 50.0 

Aqua 100.0 

Acetic  acid 10.0. — M. 

2.  Rinse  in  i  per  cent,  acetic  acid. 

3.  Alcohol  to  dehydrate.     Mount  in  balsam. 

The  capsule  will  be  found  stained  a  light  blue,  the  bacillus  a 
deep  blue.  (See  also  the  Capsule  Stain  of  Hiss,  p.  35,  and  that 
of  Burger.) 

Patho  genesis. — Animals  are  not  affected  unless  the  culture  is 
injected  intrapleural. 

Pneumobacillus  of  Frankel  (A.  Frankel  and  Weichsel- 
baum). — Synonyms. — Pneumococcus;  diplococcus  of  pneu- 
monia; micrococcus  of  sputum  septicemia;  Micrococcus 
pasteuri;  Diplococcus  lanceolatus. 

Origin. — A.  Frankel  found  it  in  the  sputum  of  pneumonic 
patients,  thinking  it  at  first  to  be  the  micrococcus  of  sputum 
septicemia;  later  he  believed  it  to  be  the  cause  of  pneu- 
monia. 

Form. — They  were  at  first  called  oval  cocci,  but  they  are  now 
known  to  be  rod-shaped,  being  somewhat  longer  than  broad, 
varying,  however,  much  in  size  and  shape.  Usually  found  in 
pairs,  sometimes  in  filaments  of  three  and  four  elements.  In 
the  material  from  the  body  a  capsule  surrounds  each  rod.  In 
the  artificial  cultures  this  is  not  found. 

Properties. — They  are  without  self -movement;  do  not  liquefy 
gelatin.  There  are  no  spores. 


152  ESSENTIALS    OF    BACTERIOLOGY 

Growth— Grow  only  at  high  temperature — 35°  C.;  are  facul- 
tative anaerobic.  The  culture-media  must  be  slightly  alkaline; 
the  growth  is  slow. 

Colonies  on  Gelatin  Plates. — Since  the  temperature  must  be 
somewhat  elevated,  the  gelatin  media  need  to  be  thicker  than 
usual  (15  per  cent,  gelatin),  in  order  to  keep  it  solid,  and  a 
temperature  of  24°  C.  used.  Little  round  white  colonies,  some- 
what granular  in  the  center,  growing  very  slowly. 

Stab-cultures. — Along  the  needle-track  small  separate  white 
granules,  one  above  the  other,  like  a  string  of  beads. 

Stroke-culture. — On  agar,  transparent,  almost  invisible  little 
drops,  resembling  dew  moisture. 


Fig.  88. — Bacillus  of  pneumonia  in  blood  of  rabbit  (x  1000)  (Frankel  and 
Pfeiffer). 

Bouillon. — They  grow  better  here  than  in  the  other  media, 
remaining  alive  a  longer  period  of  time. 

Blood-serum  and  Agar. — A  good  growth  on  blood-serum  or 
blood-agar. 

Staining. — Takes  Gram's  method  and  the  other  anilin  stains 
very  readily.  The  capsule  stained  the  same  way  as  that  of  the 
Friedldnder  bacillus. 

Patho genesis. — Rabbits  and  guinea-pigs,  if  subcutaneously 
injected,  die  in  the  course  of  a  couple  of  days  with  septicemia 
(o.i  c.c.  of  a  fresh  bouillon  culture  suffices). 


PATHOGENIC    BACTERIA  153 

Autopsy  shows  greatly  enlarged  spleen  and  myriads  of  bacilli 
in  the  blood  and  viscera,  the  lungs  not  especially  affected.  If 
injected  into  the  trachea,  a  pneumonia  occurs.  In  man  they 
are  found  in  90  per  cent,  of  croupous  pneumonia,  and  usually 
only  during  the  existence  of  the  rusty  sputum,  i.  e.,  the  first 
stage. 

The  bacilli  have  also  been  found  in  pleuritis,  peritonitis,  peri- 
carditis, meningitis,  and  endocarditis.  They  stand  in  some 
intimate  relation  to  all  infectious  inflammations  of  the  body. 
Their  presence  in  healthy  mouth  secretion  does  not  speak 
against  this,  it  requiring  some  slight  injury  to  allow  this  ever- 
present  germ  to  develop  into  disease. 

Antitoxin  of  Pneumonia  (Klemperer) . — The  injection 
of  very  diluted  cultures  of  the  virulent  bacilli  intravenously 
has  produced  an  immunity  in  rabbits  and  guinea-pigs.  The 
serum  of  such  artificially  immune  animals  when  filtered  through 
a  Chamberland  filter  and  injected  into  a  rabbit  suffering  with 
pneumonia,  cured  the  same;  or  when  injected  into  a  susceptible 
animal,  produced  in  it  immunity  very  quickly.  This  principle 
is  ascribed  to  an  antitoxin  formed  in  the  tissues  by  the  diluted 
proteids,  and  this  antitoxin  neutralizes  the  toxicity  of  the  strong 
virus.  The  attempt  to  treat  with  antitoxins  has  been  aban- 
doned, the  disease  ending  so  rapidly  by  crisis  that  it  is  difficult 
to  trace  any  curative  effect  from  remedies. 

Opsonins.-^-Tbt  opsonic  index  in  the  studies  so  far  made 
does  not  show  any  marked  change  from  the  normal. 

Virulent  pneumococci  are  insusceptible  to  phagocytosis. 

Bacillus  of  Rhinoscleroma  (Frisch,  1882).— It  was  found 
in  the  tissue  of  a  rhinoscleroma,  but  resembles  the  Friedlander 
bacillus  in  nearly  every  respect,  and  as  the  disease  rhino- 
scleroma  is  not  reproduced  by  the  inoculation  of  the  bacillus 
in  animals,  it  can  be  considered  identical.  The  growth,  cul- 
tures, and  properties  are  the  same  as  the  pneumobacillus  of 
Friedlander. 

Diplococcus  Intracellularis  Meningitidis  (Weichsel- 
baum) . — Origin. — Found  by  Weichselbaum  in  epidemic  cere- 
brospinal  meningitis  in  1887. 


154  ESSENTIALS    OF    BACTERIOLOGY 

Form. — A  small  coccus  occurring  in  pairs,  flattened  against 
each  other,  and  contained  within  the  leukocytes. 

Properties. — Ferments  sugars,  with  acid  production. 

Growth. — Best  in  blood-agar,  serum-agar,  and  ascitic  glucose- 
agar  at  body  temperature  in  twenty-four  hours. 

Colonies. — Circular  discs,  almost  transparent  under  low 
power;  margins  smooth. 


Fig.  89. — Diplococcus  intracellularis  meningitidis  in  leukocytes.  Cover- 
glass  preparation  from  peritoneal  exudate  in  a  guinea-pig  (x  2000)  (Wright 
and  Brown). 

Stain. — With  basic  anilin.  Gram  negative,  resembling 
gonococcus. 

Patho genesis. — Causes  epidemic  cerebrospinal  fever,  prob- 
ably by  infection  through  the  nasopharynx;  the  organism  is 
found  in  the  spinal  fluid  and  in  other  inflammatory  exudates, 
and  can  be  examined  in  fluid  by  lumbar  puncture. 

Ordinary  laboratory  animals  immune,  but  Flexner  has 
succeeded  in  inoculating  monkeys. 


PATHOGENIC    BACTERIA  155 

Agglutination. — On  the  fourth  day;  in  dilution  of  i  :  50 
agglutination  is  bad. 

Protective  Serum. — Flexner  has  been  able  to  obtain  an  anti- 
toxin from  monkey  serum  that  has  therapeutic  properties  in 
man. 

Micrococcus  Tetragenus  (Koch;  Gaffky). — Origin. — 
Koch  found  this  microbe  in  the  cavity  of  a  tuberculous  lung. 
Gaffky,  in  1883,  studied  its  pathogenic  actions  and  gave  it  the 
name  it  now  bears. 

Form. — Cocci  which  are  gathered  in  the  tissues  in  groups  of 
four,  forming  a  square — a  tetrad.  (See  Fig.  90.)  In  artificial 


•  ^-^r 

Fig.  90. — Micrococcus  tetragenus  in  sputum  (tubercle  bacillus  also). 

culture  sometimes  found  in  pairs.  A  capsule  of  light,  gelati- 
nous consistence  surrounds  each  tetrad. 

Properties. — They  are  immobile;  do  not  liquefy  gelatin. 

Growth. — They  grow  well  on  all  nutrient  media  at  ordinary 
and  brood  temperatures;  are  facultative  aerobic.  They  grow 
slowly. 

Colonies  in  gelatin  plates.  In  two  days,  little  white  spots, 
which,  when  on  the  surface,  form  little  elevations  of  a  porcelain- 
like  appearance;  under  low  power  they  are  seen  very  finely 
granulated. 

Stab-culture. — Small   round    separated    colonies   along    the 


156 


ESSENTIALS    OF    BACTERIOLOGY 


needle-track,  and  on  the  surface  a  button-like  elevation — a 
form  of  "nail  culture."  (See  Fig.  91.) 

Potato. — A  thick,  slimy  layer  which  can  be 
loosened  in  long  shreds. 

Staining. — Colored  with  the  ordinary  ani- 
lin  stains.  Gram's  method  also  applicable. 

Patho genesis. — White  mice  and  guinea-pigs 
die  in  a  few  days  of  septicemia  when  injected 
with  the  tetragenus  cultures,  and  the  micro- 
coccus  is  then  found  in  large  numbers  in  the 
blood  and  viscera.  Field-mice  are  immune. 

In  the  cavities  of  tuberculous  lungs,  in  the 
sputum  of  phthisical  and  healthy  patients,  it 
is  often  found,  but  what  action  it  has  upon 
man  has  not  yet  been  determined. 

Capsule  Bacillus  (Pfeiffer). — Origin. — 
Stringy  exudate  and  blood  of  a  dead  guinea- 

Form. — Thick  little  rods,  sometimes  in  long 
threads.  Large  oval  capsules  in  the  stained 
preparations  (Fig.  92). 

Properties. — Immotile,  not  liquefying,  an 
odorless  gas  in  gelatin  cultures. 

Growth. — At  ordinary  temperatures,  rap- 
idly; facultative  anaerobin. 

Gelatin  Plates. — Oval  points,  and  like  a  por- 
celain button  on  the  surface. 

Stab-cultures. — Like  the  pneumonia  ba- 
cillus of  Friedlander. 

Potato. — Abundant  growth,  yellow  color  and  moist,  coming 
off  in  strings. 

Staining. — Hot  fuchsin  colors  the  capsule  intensely;  carefully 
decolorizing  with  acetic  acid,  the  capsules  are  red  or  light  violet 
around  the  deeply  tinged  bacillus.  Gram's  method  not  applic- 
able. 

Pathogenesis. — Subcutaneously  injected  in  mice,  they  die  in 
forty-eight  hours.  Rabbits  die  when  a  large  quantity  is  injected 


Fig.  91.  —  Stab- 
culture.  Micrococ- 
cus  tetragenus. 


PATHOGENIC   BACTERIA  157 

into  the  circulation.     The  blood  and  juices  have  a  peculiar 
stringy,  fibrinous  consistence. 


Fig.  92. — Pfeiffer's  capsule  bacillus  in  blood  (X  1000)  (Friinkel  and  Pfeiffer). 

Bacillus  of  Influenza  (Pfeiffer,  18Q2).— Origin.— A  small 
bacillus,  about  one-half  the  size  of  the  bacillus  of  mouse  septi- 
cemia,  and  arranged  in  chain  form.  It  develops  upon  blood- 
serum  agar.  It  is  aerobic,  without  movement;  does  not  take 
the  Gram  stain  (Fig.  93). 

Stain, — It  is  best  stained  with  diluted  carbol-fuchsin,  the 
contrast-stain  being  Loffler's  methylene-blue. 

Growth. — Upon  glycerin-agar,  over  which  a  drop  of  blood 
has  been  spread,  in  an  incubator  at  the  end  of  twenty-four 
hours,  a  very  delicate  growth  occurs,  which  resembles  condensed 
moisture. 

Pathogenesis. — It  is  found  in  the  sputum  and  in  the  bron- 
chial nasal  secretions  and  blood  of  influenza  patients.  It  has 
been  transmitted  to  monkeys;  other  animals  are  not  suscep- 
tible. It  has  never  been  found  outside  the  body.  Its  resist- 
ance is  very  feeble;  in  water,  the  bacilli  die  in  twenty-four 


158  ESSENTIALS    OF    BACTERIOLOGY 

hours,  but  sputa  containing  the  germs  may  be  ejected  for  days 
and  weeks.     Influenza  bacilli  are  found  accompanying  bron- 


•• 


*  <**  >i.4«yx          v* 

v^7'^'"    **<^     v&  '.£• 
k5-  *  «*f- 

^rV/> 

!f-l?^:    vv     •   k 

»   *v*'?*    *.  *-  :••  :cJV^ 
*•*»••  *  \«  *' 


Fig.  93.  —  Bacillus  influenzae,  from  a  gelatin  culture  (x  1000)  (Itzerott  and 
Niemann). 

chopneumonia,  tuberculosis,  meningitis,  and  other  inflam- 
mations. 

Microorganisms  of  Suppuration.  —  The  suppuration  of 
wounds  is  due  to  the  presence  of  germs.  The  knowledge  of 
this  fact  is  the  basis  of  the  antiseptic  treatment  in  surgery; 
for  when  the  microbes  can  be  destroyed  or  their  entrance  pre- 
vented, the  wounds  are  made  clean  and  kept  without  suppurat- 
ing. Various  forms  of  bacteria  have  been  found  in  septic 
processes,  and  the  formation  of  pus  cannot  be  ascribed  to 
any  particular  one  alone;  some,  more  common  than  others, 
are  found  in  nearly  all  forms  of  suppuration;  others  give  rise 
to  special  types. 

Wounds  are  often  irritated  by  foreign  bodies  and  chemicals, 
and  a  discharge  occurs  in  them  even  when  every  aseptic  and 
antiseptic  precaution  has  been  taken;  but  such  a  discharge  is 
free  from  bacteria,  and  no  more  like  pus  than  a  benign  growth 
is  like  a  malignant  one. 


PATHOGENIC   BACTERIA 


159 


Streptococcus  Pyogenes  (Rosenbach) ;  Streptococcus 
erysipelatis  (Fehleisen) . — Origin. — Fehleisen  discovered  this 
microbe  in  the  lymphatics  of  the  skin  in  erysipelas,  and  he 
thought  it  the  cause  of  the  same.  Under  the  name  Streptococcus 
pyogenes,  Rosenbach  described  an  identical  coccus  which  has 
been  found  in  nearly  all  suppurative 
conditions. 

Form. — Small  cocci  singly  and  in 
chain-like  groups.  Spores  have  not 
been  found,  though  it  is  supposed, 
because  of  their  permanency,  that 
spores  are  present. 

Properties. — They  are  immotile;  do 
not  liquefy  gelatin. 


Fig.  94. — Streptococcus 
pyogenes:  culture  upon  agar- 
agar  two  days  old  (Frankel 
and  Pfeiffer). 


Fig.  95. — Streptococcus  pyogenes 
(Jakob). 


Growth. — They  grow  slowly,  usually  on  the  surface,  and  best 
at  higher  temperatures. 

Colonies. — In  three  days  a  very  small  grayish  speck,  which 
hardly  ever  becomes  much  larger  than  a  pin-head;  under 
microscope,  looking  yellowish,  finely  granular,  the  edges  well 
defined. 

Stab-cidtures . — Along  the  needle-track  little  separated  col- 


l6o  ESSENTIALS    OF    BACTERIOLOGY 

onies,  like  strings  of  beads,  which  after  a  time  become  one  solid 
white  string. 

Stroke-culture  on  Agar. — Little  drops,  never  coalescing,  hav- 
ing a  bluish  tint,  very  transparent. 

Potato. — No  apparent  growth. 

Bouillon. — At  37°  C.  clouds  are  formed  in  the  bouillon, 
which' then  sink  to  the  bottom,  and  long  chains  of  cocci  found 
in  this  growth. 

Staining. — Easily  colored  with  the  ordinary  stains.  Gram's 
method  is  also  applicable. 

Patho  genesis. — Inoculated  subcutaneously  in  the  ear  of  a 
rabbit,  an  erysipelatous  condition  develops  in  a  few  days, 
rapidly  spreading  from  point  of  infection. 

In  man,  inoculations  have  been  made  to  produce  an  effect 
upon  carcinomatous  growths  and  erysipelas  was  always  pro- 
duced. When  it  occurs  upon  the  valves  of  the  heart,  endo- 
carditis results.  Puerperal  fever  is  caused  by  the  microbe 
infecting  the  endometrium,  the  Streptococcus  puerperalis  of 
Frankel  being  the  same  germ. 

In  scarlatina,  variola,  yellow  fever,  cerebrospinal  meningitis, 
and  many  similar  diseases,  the  microbe  has  been  an  almost  con- 
stant attendant.  It  is  often  associated  with  the  diphtheria 
bacillus  in  true  diphtheria,  and  is  the  cause  of  many  of  the 
diphtheritic  affections  of  the  throat  in  which  the  diphtheria 
bacillus  is  absent. 

An  antistreptococcic  serum  has  been  used  as  a  curative 
agent  in  puerperal  fever,  scarlatina,  and  other  diseases  sup- 
posed to  be  due  to  this  germ. 

A  mixture  of  a  culture  of  pyogenes  and  prodigiosus  has  been 
used  as  an  injection,  with  apparent  benefit,  in  inoperable  cases 
of  sarcoma,  and  is  known  as  Coley's  fluid. 

Staphylococcus  Pyogenes  Aureus  (Rosenbach) . — Origin. 
— Found  commonly  in  pus  (80  per  cent,  of  all  suppura- 
tions), in  air,  water,  and  earth;  also  in  sputum  of  healthy 
persons. 

Form. — Micrococci  in  clusters  like  bunched  grapes,  hence  the 
name  staphylo,  which  means  grape.  They  never  form  chains. 


PATHOGENIC    BACTERIA  l6l 

Spores  have  not  been  found,  though  the  cocci  are  very 
resistant. 

Properties. — Without  movement;  liquefying  gelatin.  It  gives 
rise  to  an  orange-yellow  pigment  in  the  various  cultures. 

Growth. — It  grows  moderately  fast  at  ordinary  temperature, 
and  can  live  without  air,  a  facultative  aerobin  and  anaerobin. 

Colonies  on  Gelatin. — On  second  day  small  dots  on  the  sur- 
face, containing  in  their  center  an  orange-yellow  spot.  The 
gelatin  all  around  the  colony  is  liquefied;  the  size  is  never  much 
greater  than  that  attained  the  second  day. 


Fig.  96. — Staphylococcus  pyogenes  albus  (Jakob). 

Colonies  on  A  gar. — The  pigment  remains  for  a  long 
time. 

Stab-culture. — At  first,  gray  growth  along  the  track,  which, 
after  three  days,  has  settled  at  the  bottom  of  the  tube  in  a  yel- 
low, granular  mass,  the  gelatin  being  all  liquid. 

Stroke-culture  on  Agar. — The  pigment  diffused  over  the  sur- 
face where  the  growth  is,  in  moist  masses. 

Potato. — A  thin  white  layer  which  gradually  becomes  yellow 
and  give  out  a  doughy  smell. 

Staining. — Very  readily  colored  with  ordinary  stains;  also 
with  Gram's  method. 


162 


ESSENTIALS    OF    BACTERIOLOGY 


Patho genesis. — When  rabbits  are  injected  with  cultures  of 
this  microbe  into  the  knee-joint  or  pleura,  they  die  in  a  day. 
If  injected  subcutaneously,  only  a  local  action  occurs,  namely, 
abscesses. 

If  directly  into  circulation,  a  general  phleg- 
monous  condition  arises,  the  capillaries  be- 
come plugged  with  masses  of  cocci,  infarcts 
occur  in  kidney  and  liver,  and  metastatic 
abscesses  form  in  viscera  and  joints.  Garre, 
by  rubbing  the  culture  on  his  forearm,  caused 
carbuncles  to  appear. 

Several  varieties  of  the  pyogenic  staphylo- 
cocci  are  recognized  according  to  their  color- 
producing  properties  and  slight  variations  of 
growth.     Of  these,  the  Staphylococcus  pyo- 
genes  aureus  is  the  most  virulent,  and  is  con- 
sidered the  type  of  the  group.     They  are 
always  present  on  the  surface  of  the  body, 
beneath  the  nails,  in  the  nose  and  mouth,  in 
the  dust  of  streets,  and  on  the  floor  of  houses. 
Staphylococcus  pyogenes  albus  differs 
from  the  preceding  only  in  the  absence  of 
pigment  and  in  its  slight  virulence.     Welch 
describes  a  variety  constantly  found  both  on  the  skin  and  in 
its  deeper  layers,  which  he  calls  the  Staphylococcus  epidermidis 
albus. 

Micrococcus  Pyogenes  Citreus  (Passet) .— This  liquefies 
gelatin  less  rapidly  than  the  pyogenes  aureus,  and  forms  a 
citron-yellow  pigment  instead  of  the  orange-yellow  of  the  aureus. 
Micrococcus  Cereus  Albus  (Passet)  .—Differs  from  the 
pyogenes  albus  in  the  form  of  colony.  A  white,  shiny  growth, 
like  drops  of  wax;  hence  the  name,  cereus. 

Micrococcus  Cereus  Flavus  (Passet).— A  lemon-yellow 
colored  growth  after  a  short  time,  otherwise  not  differing  from 
cereus  albus. 

Micrococcus  Pyogenes  Tenuis  (Rosenbach) .— Origin.— 
Found  in  the  pus  of  large  inclosed  abscesses. 


Fig.  97.— Stab- 
culture.  Micro- 
coccus  pyogenes 
aureus. 


PATHOGENIC   BACTERIA  163 

Form. — Cocci,  without  any  especial  arrangement. 

Properties. — Not  much  studied. 

Growth. — Cultivated  on  agar,  it  forms  clear,  thin  colonies; 
along  the  needle-track  an  opaque  streak,  looking  as  if  varnished 
over. 

Bacillus  Pyocyaneus  (Gessard) . — Synonyms. — Bacterium 
aeruginosum;  Bacillus  fluorescens  (Schroter);  the  bacillus  of 
bluish-green  pus. 

Origin. — Found  in  1882  in  green  pus  in  pyocyemia. 


Fig.  98. — Bacillus  pyocyaneus,  from  an  agar-agar  culture  (x  1000)  (Itzerott 
and  Niemann). 

Form. — Small  slender  rods  with  rounded  ends,  easily 
mistaken  for  cocci.  Often  in  groups  of  four  and  six,  without 
spores. 

Properties. — Very  motile;  liquefy  gelatin  rapidly;  a  peculiar 
sweetish  odor  and  a  blue  pigment  are  produced  in  the  cultures. 

Growth. — Develops  readily  at  ordinary  temperature,  growing 
quickly  and  mostly  on  the  surface;  it  is  aerobic.  Colonies  on 
gelatin  plate:  In  two  or  three  days  a  greenish  iridescence  appears 
over  the  whole  plate,  the  colonies  having  a  funnel-shaped  lique- 


164  ESSENTIALS    OF    BACTERIOLOGY 

faction,  and  appearing,  under  low  power,  when  still  young,  as 
yellowish  green,  the  periphery  being  granulated. 

On  agar:  A  bright  green  at  first,  causing  fluorescence;  then 
later  a  blue  pigment. 

Stab-cultures. — Mainly  in  upper  strata,  the  liquefaction 
funnel  shaped,  the  growth  gradually  settling  at  the  bottom,  a 
rich  green  shimmer  forming  on  the  surface,  and  the  gelatin 
having  a  deep  fluorescence. 

Potato. — The  potato  is  soaked  with  the  pigment,  a  deep  fold 
of  green  occurring  on  the  surface. 


Fig.  99. — Koch-Weeks'  bacillus  from  cpnjunctival  exudate   at   third  day  of 
epidemic  conjunctivitis  (Boston). 

Staining. — With  ordinary  anilin  dyes. 

Pathogenesis. — When  animals  are  injected  with  fresh  cultures 
in  the  peritoneal  cavities  or  cellular  tissues,  a  rapidly  spreading 
edema  with  general  suppuration  develops.  The  bacilli  are 
found  in  the  viscera  and  blood. 

If  a  small  quantity  is  injected,  a  local  suppuration  occurs,  and 
if  the  animal  does  not  die,  it  then  can  withstand  large  quanti- 
ties. It  is  immune. 

The  Pigment. — Pyocyanin. — When  the  pus,  bandages,  and 


PATHOGENIC   BACTERIA  165 

dressings  containing  the  Bacillus  pyocyaneus  are  washed  in 
chloroform,  the  pigment  is  dissolved  and  crystallizes  from  the 
chloroform  in  long  needles.  It  is  soluble  in  acidulated  water, 
which  is  turned  red  thereby,  and  when  neutralized,  the  blue  color 
returns.  It  has  no  pathogenic  action.  It  is  an  aromatic  com- 
pound. The  bacillus  has  no  especial  action  on  the  wound,  and 
is  found  sometimes  in  perspiration  of  healthy  persons. 

Bacillus   Pyocyaneus  /?   (Ernst). — A  bacillus   found   in 
grayish,  pus-colored  bandages. 


Fig.  ioo. — Morax-Axenfeld  diplobacillus  from  conjunctival  exudate  during 
course  of  subacute  conjunctivitis  (obj.  B.  and  L.,  one-twelfth  oil-immersion) 
(Boston). 

The  only  especial  difference  between  this  and  the  above  is  the 
formation  of  brownish-yellow  pigment  instead  of  pyocyanin. 
The  form  and  appearance  of  cultures  otherwise  the  same. 

Koch-Weeks'  Bacillus  (1883-87).— Cause  of  epidemic 
conjunctivitis,  or  "pink  eye";  found  in  the  secretion. 

Form. — Very  minute  bacillus,  resembling  the  influenza 
bacillus;  non-motile.  (See  Fig.  99.) 

Growth. — They  grow  best  on  blood-serum  agar,  but  very 
sparsely  in  minute  transparent  colonies;  non-liquefying. 


1 66  ESSENTIALS    OF    BACTERIOLOGY 

Stains. — With  carbol-fuchsin,  and  is  often  intracellular. 
Does  not  take  Gram. 

Patho genesis. — Very  contagious,  found  in  10  per  cent,  to  20 
per  cent,  of  all  cases  of  conjunctivitis.  Not  infectious  for  lower 
animals,  and  not  causing  any  other  form  of  disease. 

Morax-Axenfeld  Diplobacillus  of  Conjunctivitis. — 
This  bacillus  is  found  in  the  greater  number  of  cases  of  con- 
junctivitis. 

Form. — A  short,  plump  bacillus,  usually  in  pairs  and  chains 
of  pairs.  Non-motile  (Fig.  100). 

Growth. — With  difficulty  in  blood-serum  agar,  it  forms  small 
pitted  colonies  or  lacunae ;  liquefies. 

Staining. — Does  not  take  Gram,  but  stains  readily. 
Non-pathogenic  for  lower  animals. 

Micrococcus  Gonorrhoeas   (Gonococcus)    (Neisser). — In 

1879  Neisser  demonstrated  the  presence  of 

this  germ  in  the  secretion  of  specific  urethritis. 

,£  Form. — Cocci,     somewhat    triangular    in 

form,  found  nearly  always  in  pairs,  the  base 

of  one  coccus  facing  the  base  of  the  other 

f     v .  and  giving  the  appearance  of  a  Vienna  roll, 

hence  the  German  name,  semmel  (roll),  form. 

Four  to  twelve  such  pairs  are  often  found 

together.     Immotile  (Fig.  102). 

GonFocgocci  in°go^         Culture.— On  gelatin-agar  or  potato  they 
orrheaipus.    Ani-      do  not  grow,  and  onlv  upon  human  blood- 

lin      methyl-violet  '  .  ( 

(x  650).  serum  have  they  given  any  semblance  of  a 

growth.  The  temperature  must  be  be- 
tween 33°  and  37°  C.,  and  the  growth  occurs  very  slowly  and 
sparsely. 

Method  of  Cultivation  (Wertheini) . — Gonorrheal  pus  is  mixed 
in  a  test- tube  with  liquid  human  blood-serum  of  40°  C.  temper- 
ature, and  two  dilutions  are  made  with  blood  of  the  same  tem- 
perature. An  equal  quantity  of  2  per  cent,  agar  solution  is 
now  poured  into  each  tube,  and  three  glass  dishes  are  covered 
at  once  with  this  mixture.  After  being  in  the  brood-oven  for 
twenty-four  hours,  colonies  can  be  discovered. 


PATHOGENIC   BACTERIA  167 

In  three  days  a  very  thin,  almost  invisible,  moist  yellowish 
growth,  seemingly  composed  of  little  drops. 

Under  low  power  small  processes  are  seen  shooting  out  from 
the  smooth  border. 

It  requires  then  to  be  transferred  to  fresh  media,  as  the  cul- 
ture quickly  perishes. 

Cultivation  has  also  occurred  on  acid  gelatin,  gelatin  con- 
taining acid  urine,  in  acid  urine  itself,  and  albuminous  urine 
with  agar. 


Fig.  102. — Gonococcus  in  urethral  pus  (x  1000)  (Frankel  and  Pfeiffer). 

In  septicemic  cases  the  gonococcus  has  been  isolated  from 
the  blood  direct  by  drawing  5  to  10  c.c.  from  a  vein  and  adding 
it  in  equal  parts  to  melted  agar.  The  mixture  is  poured  into 
Petri  dishes  and  developed  in  the  incubator  at  37°  C. 

Staining. — Colored  easily  with  all  ordinary  anilin  stains. 

Gram's  method  is  not  applicable,  this  being  one  of  its  main 
diagnostic  features. 

The  following  method  is  recommended  by  Neisser. 

The  cover-glasses,  with  some  of  the  urethral  discharge 
smeared  upon  them,  are  covered  with  a  few  drops  of  alcoholic 


1 68  ESSENTIALS    OF    BACTERIOLOGY 

solution  of  eosin,  and  heated  for  a  few  minutes  over  the  flame. 
The  excess  of  the  dye  is  removed  with  filter-paper,  then  the 
cover-glass  placed  in  concentrated  methylene-blue  (alcoholic 
solution)  for  fifteen  seconds,  and  rinsed  in  water. 

The  gonococci  are  dark  blue,  the  protoplasm  of  the  cell  pink, 
and  the  nucleus  a  light  blue,  the  gonococci  lying  in  the  proto- 
plasm next  to  the  nucleus  (Fig.  101). 

Other  bacteria  are  similar  to  the  gonococci  in  form;  they  are 
distinguished  from  the  gonococcus  in  that  they  are  positive 
with  Gram's  method,  whereas  the  micrococcus  of  gonorrhea  is 
not.  The  points  on  which  the  diagnosis  is  to  be  made  are  the 
characteristic  biscuit  shape,  the  intracellular  position  of  the 
organism,  and  its  failure  to  stain  with  Gram. 

Patho  genesis. — The  attempts  to  infect  the  experiment  ani- 
mals with  gonorrhea  have  so  far  been  without  success.  In 
man,  upon  a  healthy  urethra,  a  specific  urethritis  was  produced 
with  even  the  twentieth  generation  of  the  culture.  Gonorrheal 
ophthalmia  contains  the  cocci  in  great  numbers,  and  endocarditis 
and  gonorrheal  rheumatism  are  said  to  be  caused  by  the  cocci. 

The  micrococci  have  been  found  long  after  the  acute  attack, 
when  only  a  very  slight  oozing  remained,  and  the  same  were 
very  virulent. 

The  specific  inflammations  of  the  generative  organs  of  the 
female  are  due  to  this  organism  gaining  entrance  through  the 
vagina.  It  is  found  chiefly  in  the  superficial  layers  of  the 
mucous  membrane. 

A  temperature  of  40°  C.  for  twelve  hours  destroys  the 
gonococci. 

Gonotoxin. — A  toxin  has  been  isolated  which  causes  fever, 
loss  of  weight,  and  finally  death .  The  urethra  is  not  immunized 
by  repeated  injections.  In  man  the  toxin  causes  painful 
indurations,  lasting  several  days. 

Similar  Bacteria  Found  in  the  Urethra  and  Vagina.— 
Micrococcus  Citreus  Conglomerata  (Bumm)  .—Similar  to 
the  gonococci  in  form,  they  are,  however,  easily  cultivated,  and 
form  yellow  colonies  which  dissolve  the  gelatin  and  grow  rap- 
idly; the  surface  of  the  gelatin  is  at  first  moist  and  shiny,  but 


PATHOGENIC   BACTERIA  169 

later  on  wrinkled.  Colored  with  Gram's  method,  and  have  no 
special  pathologic  action.  Found  in  the  air  and  gonorrheal  pus. 

Diplococcus  Albicans  Amplus  (Bumm).— In  vaginal 
secretion.  The  diplococci  are  much  larger  than  the  gonococci, 
but  similar  in  form.  They  are  also  cultivated  upon  gelatin 
plates — grayish-white  colonies,  which  slowly  liquefy  gelatin. 
They  grow  moderately  rapid.  Stained  with  Gram's  method, 
and  have  no  pathogenic  action. 

Diplococcus  Albicans  Tardissimus  (Bumm). — Origin. — 
In  urethral  pus.  Form,  like  gonococci.  Properties,  immotile; 
do  not  liquefy  gelatin.  Growth,  very  slow  at  ordinary  tem- 
perature, but  more  rapid  at  brood-heat.  The  colonies  show 
small  white  points,  which  under  low  power  appear  brown 
and  opaque. 

Agar  Stroke-culture. — Grayish-white  growth,  which  after 
two  months  is  like  a  skin  upon  the  surface. 

Staining. — Takes  Gram's  method. 

Patho genesis. — None  known. 

Micrococcus  Subflavus  (Bumm). — Origin. — In  lochial 
discharges,  in  vagina  and  urethra  of  healthy  persons. 

Form. — As  gonococci. 

Properties. — Not  motile;  liquefy  gelatin  slowly;  a  yellow- 
brownish  pigment. 

Growth. — Grows  slowly  on  all  media,  forming  on  gelatin, 
after  two  weeks,  a  moist  yellowish  surface  growth. 

Potato. — Small  half-moon-shaped  colonies  which,  after  three 
weeks,  become  light  brown  in  color,  and  covering  the  surface  as 
a  skin. 

Staining. — Colored  with  Gram. 

Pathogenesis. — Not  acting  upon  the  mucous  membrane,  but 
when  injected  in  cellular  connective  tissue,  an  abscess  results 
which  contains  myriads  of  diplococci. 

The  gonococcus  is  distinguished  from  all  these  similar  micro- 
cocci  by — 

First. — Being  found  usually  within  the  cell  protoplasm. 

Second. — Not  stained  with  Gram's  method. 

Third. — Refusing  to  grow  readily  upon  gelatin. 


1 70         ESSENTIALS  OF  BACTERIOLOGY 

All  the  similar  bacteria  being  easily  cultivated. 

These  characteristics,  taken  in  toto,  form  sufficient  features 
for  its  ready  recognition,  and  as  it  is  often  a  serious  question  to 
decide,  not  so  much  because  of  the  patient's  health  as  because 
of  his  character,  we  should  be  very  careful  not  to  pronounce  a 
verdict  until  we  have  tested  the  microorganism  as  above.  When 
the  germ  is  found  which  answers  to  the  above  description,  the 
process  can  be  called  specific  without  a  doubt. 

Protective  Vaccines  in  Pus  Infections.— Wright  has  used 
the  opsonic  treatment  in  infections  due  to  pus-forming  organ- 
isms with  most  marked  success. 

Autovaccines  are  made  by  using  cultures  of  the  lesion.  An 
agar  slant  is  obtained,  and  the  growth  washed  in  normal  salt 
solution  and  killed  by  steam.  The  dead  bacteria  in  a  cubic 
centimeter,  computed  according  to  Wright's  method  (which 
see),  and  known  quantities  of  bacteria — from  250,000,000  to 
500,000,000  dead  cells — are  injected  into  the  patient. 

The  opsonic  index  is  first  estimated,  and  then  measured 
every  few  days,  giving  the  injection  during  the  positive  phase. 
If  autovaccines  cannot  be  prepared,  laboratory  cultures  of 
mixed  germs  can  be  used.  In  boils,  acne  pustules,  gonococcus 
infections,  and  Bacillus  coli  abscesses  good  results  have  been 
reported. 

Bacillus  of  Tetanus  (Nicolaier-Kitasato) . — Origin. — 
Nicolaier  found  this  bacillus  in  the  pus  of  a  wound  in  one 
who  had  died  of  tetanus,  describing  it  in  1884. 

Kitasato  isolated  and  cultivated  this  germ  (1889). 

Form. — A  very  delicate,  slender  rod,  somewhat  longer  than 
the  bacillus  of  mouse  septicemia,  which  is  the  smallest  bacillus 
known. 

When  the  spores  form,  a  small  swelling  occurs  at  the  end 
where  the  spore  lies,  giving  it  a  drum-stick  shape. 

Properties. — Not  very  motile,  though  distinctly  so;  liquefies 
gelatin  slowly.  The  cultures  give  rise  to  a  foul-smelling  gas. 

Growth. — Develops  very  slowly,  best  at  brood-heat  (36°  to  38° 
C.) ,  and  only  when  all  oxygen  is  excluded — an  obligatory  anaero- 
bin.  In  an  atmosphere  of  carbon  dioxid  gas  it  cannot  grow, 
but  in  hydrogen  it  flourishes. 


PATHOGENIC   BACTERIA  171 

Colonies  on  gelatin  plates  in  an  atmosphere  of  hydrogen. 
Small  colonies.  After  four  days  a  thick  center  and  radiating 
wreath-like  periphery,  like  the  colonies  of  Bacillus  subtilis. 

High  Stab-culture. — The  gelatin  having  2  per  cent,  glucose 
added  and  filling  the  tube.  Along  the  lower  portion  of  the 
needle -track,  a  thorn-like  growth,  little  needle-like  points 
shooting  out  from  a  straight  line.  The  whole  tube  becomes 


Fig.  103. — Bacillus  of  tetanus  with  spores. 

clouded  as  the  gelatin  liquefies,  and  then  the  growth  settles  at 
the  bottom  of  the  tube. 

Agar. — At  brood-heat,  on  agar,  the  growth  is  quite  rapid,  and 
at  the  end  of  forty-eight  hours  gas-bubbles  have  formed  and  the 
growth  nearly  reached  the  surface. 

Bouillon. — Adding  glucose  to  the  bouillon  gives  a  medium  in 
which  an  abundant  growth  occurs. 

Cultivation  from  Spores. — Kitasato,  by  exposing  a  portion  of 
suspected  material  to  a  temperature  of  80°  C.  for  one  hour, 
killed  off  all  the  spores  save  those  of  tetanus,  which  were  then 
cultivated. 


172 


ESSENTIALS    OF    BACTERIOLOGY 


Staining. — All  the  ordinary  stains,  Gram's  method  also,  the 
spores  being  colored  in  the  usual  way. 

Pathogenesis . — A  small  amount  of  the  pure  culture  injected 
under  the  skin  of  experiment  animals  will  cause,  in  two  to  three 


Fig.  104. — Bacillus  tetani:  cul- 
ture four  days  old  in  glucose-gela- 
tin (Frankel  and  Pfeiffer). 


Fig.  105. — Six  days'  culture  of 
bacillus  of  tetanus  in  gelatin  (deep 
stab)  (Frankel  and  Pfeiffer). 


days,  death  from  true  tetanus,  the  tetanic  condition  starting 
from  the  point  of  infection.  At  the  autopsy  nothing  charac- 
teristic or  abnormal  is  found,  and  the  bacilli  have  disappeared, 
except  near  the  point  of  entrance.  This  fact  is  explained  as 
follows : 


PATHOGENIC    BACTERIA  173 

Several  toxic  products  have  been  obtained  from  the  cultures, 
and  they  are  produced  in  the  body,  and  give  rise  to  the  morbid 
symptoms.  These  have  been  isolated,  and  when  injected  singly, 
cause  some  of  the  tetanic  symptoms.  The  virus  enters  the 
circulation,  but  does  not  remain  in  the  tissues.  The  spores  are 
very  resistant  to  heat,  drying,  and  chemicals. 

The  blood  and  the  urine  contain  the  toxin  and  are  fatal  to 
animals. 

The  toxin  is  most  virulent.  It  acts  on  the  end-plates  of  the 
muscles,  and  then  on  the  motor  nerve-cells.  The  incubation 
period  is  from  two  to  fourteen  days  after  receipt  of  injury. 

Immunity. — Kitasato,  by  inoculation  of  sterilized  cultures, 
has  caused  immunity  to  the  effects  of  virulent  bacilli. 

An  antitoxin  obtained  by  Tizzoni  and  Cattani  from  the 
serum  of  animals  made  immune  by  sterilized  cultures  is 
used  with  curative  effects  in  cases  of  tetanus  in  man.  It  is 
a  globulin,  but  differs  from  the  anthrax  antitoxin,  and  it  is 
found  exclusively  in  the  serum.  By  precipitation  with  alcohol 
and  drying  in  vacuo  the  antitoxin  is  obtained  in  a  solid  state. 
The  aqueous  solution  is  used  for  injection  subcutaneously  or 
subdurally  through  a  trephine  opening.  Its  injection  into  the 
spinal  canal  by  lumbar  puncture  has  also  been  recommended. 
Antitoxin  is  more  beneficial  in  chronic  cases  than  in  acute. 

The  dried  antitoxin  has  been  spread  on  the  wound  with 
some  curative  action. 

The  antitetanic  serum,  to  be  effective,  must  be  given  very 
early  and  in  large  doses.  Its  greatest  use  is  in  preventing 
tetanus  in  wounds  liable  to  be  infected.  From  50  c.c.  to  100  c.c. 
of  a  billion-unit  serum  should  be  given  in  divided  doses;  only 
serums  with  very  high  protective  powers  should  be  used. 

Habitat. — The  bacillus  is  present  in  garden-earth,  in  manure, 
and  it  has  been  isolated  even  from  mortar. 

The  earth  of  special  districts  seems  to  contain  the  bacilli  in 
greater  quantities. 

Spores  of  tetanus  may  gain  access  to  animal  serums,  and 
if  not  properly  destroyed,  may  produce  tetanus  during  the  use 
of  these  products.  Great  care  and  previous  testing  should 


174 


ESSENTIALS    OF    BACTERIOLOGY 


be  used  in  the  manufacture  of  all  animal  vaccines,  antitoxins, 
etc. 

Bacillus  (Edematis  Maligni  (Koch,  1881) ;  Vibrion 
Septique  (Pasteur,  1875). — Origin. — In  garden-earth,  found 
also  in  man,  in  severe  wounds  when  gangrene  with  edema 
had  developed.  Identical  with  the  bacillus  found  in  Pasteur's 
septicemia. 

Form. — Rods  somewhat  smaller  than  the  anthrax  bacilli,  the 
ends  rounded  very  sharply.  Long  threads  are  formed.  Very 
large  spores  which  cause  the  rods  to  become  spindle  shaped. 


Fig.   106. — Bacillus  of  malignant  edema,  from  the  body-juice  of  a  guinea-pig 
inoculated  with  garden-earth  (x  1000)  (Frankel  and  Pfeiffer). 

Properties. — Very  motile;  liquefy  gelatin;  do  not  produce 
any  foul  gaseous  products  in  the  body. 

Growth. — Grows  rapidly,  but  only  when  the  air  is  excluded, 
and  best  at  brood  or  body  heat. 

Roll  Cultures  (After  Esmarch's  Method) . — Small,  round  colo- 
nies with  fluid  contents,  under  low  power,  a  mass  of  motile 
threads  in  the  center,  and  at  the  edges  a  wreath-like  border. 

High  Slab-culture. — With  glucose  gelatin,  the  growth  at  first 


PATHOGENIC   BACTERIA 


175 


seen  in  the  bottom  of  the  tube,  with  a  general  liquefaction  of 
the  gelatin;  gases  develop  and  a  somewhat  unpleasant  odor. 


I 


/ 


Fig.  107. — Cultures  in  agar 
of  malignant  edema,  after 
twenty-four  hours,  at  37°  C. 
(Frankel  and  Pfeiffer). 


Fig.  108. — Bacillus  of 
malignant  edema  growing  in 
glucose  -  gelatin  (Frankel  and 
Pfeiffer). 


Agar. — The  gases  develop  more  strongly  in  this  medium,  and 
the  odor  is  more  prominent. 

Guinea-pig  Bouillon. — In  an  atmosphere  of  hydrogen  cloud- 
ing of  the  entire  culture-medium  without  any  flocculent  pre- 
cipitate until  third  day. 


176  ESSENTIALS    OF    BACTERIOLOGY 

Staining. — Are  stained  with  the  ordinary  dyes,  but  Gram's 
method  negative. 

Pathogenesis. — When  experiment  animals,  mice  or  guinea- 
pigs,  are  injected  with  a  pure  culture  under  the  skin,  they  die  in 
eight  to  fifteen  hours,  and  the  following  picture  presents  itself 
at  the  autopsy:  In  guinea-pigs  from  the  point  of  infection, 
spreading  over  a  large  area,  an  edema  of  the  subcutaneous 
tissues  and  muscles,  which  are  saturated  with  a  clear  red  serous 
exudate,  free  from  smell,  containing  great  quantities  of  bacilli. 


Fig.  109. — Smear  of  pus  of  chancroid  of  penis   (x  !5°o)  (Davis)  (photo- 
micrograph by  Mr.  L.  S.  Brown). 

The  spleen  is  enlarged,  especially  in  mice.  The  bacilli  are 
not  found  in  the  viscera,  but  are  present  in  great  numbers  on 
the  surface,  i.  e.,  in  the  serous  coverings  of  the  different  organs; 
though  when  any  length  of  time  has  elapsed  between  the  death 
of  the  animal  and  the  examination,  they  can  be  found  in  the 
inner  portions  of  the  organs,  for  they  grow  well  upon  the  dead 
body.  In  man  they  have  been  found  in  rapidly  spreading  gan- 
grene. They  are  present  in  the  soil,  in  putrefactions  of  various 
kinds,  and  in  dirty  water. 

Immunity. — Is  produced  by  injection  of  the  sterilized  cul- 
tures, and  also  the  filtered  bloody  serum  of  animals  dead  with 
the  disease. 


PATHOGENIC   BACTERIA  177 

Bacillus  of  Soft  Chancre  (Ducrey-Unna,  188g).— Adiplo- 
bacillus  which  is  specific  has  been  described  by  Ducrey  as 
obtained  from  the  secretion  and  in  the  depth  and  margins  of 
the  chancroid.  Unna's  bacillus  is  narrower  and  unbroken  in 
the  center. 

Cultivation. — Cultivation  has  occurred  on  blood-agar,  the 
blood  being  added  in  the  proportion  of  one  to  two.  Colonies  are 
small,  round  globules. 

Staining. — With  borax,  methylene-blua,  decolorized  with 
weak  acetic  acid. 

Pathogenesis. — Probably  a  mixed  infection  occurs  in  most 
chancroids,  especially  if  buboes  result.  The  bacillus  of  Ducrey 
is  not  found  in  unopened  buboes,  though  often  contaminating 
the  ulcerated  ones. 

The  disease  has  been  reproduced  by  inoculation  of  the  human 
subject.  Laboratory  animals  are  immune . 

Bacillus  of  Bubonic  Plague  (Yersin  and  Kitasato,  1894). 
— Bubonic  plague  or  pest  is  an  extremely  infectious  disease, 


*  * 


I    £%??«*  £    , 

m$^^3 


t;^c*V>Ew 

f«'  -\»'-'Ji'  '^  . 

\;^;;->^^; 


Fig.  no. — Bacillus  of  bubonic  plague  (Yersin). 

more  or  less  common  in  China  and  the  East,  and  is  believed  to 
have  its  origin  in  man  from  rats  and  other  rodents.  It  spreads 
with  great  rapidity,  especially  among  those  living  under  unsani- 
tary conditions. 


178  ESSENTIALS    OF    BACTERIOLOGY 

Nearly  at  the  same  time  Yersin  and  Kitasato,  working  inde- 
pendently, discovered  in  the  bubonic  swellings  and  blood  of 
affected  persons  a  distinctive  bacillus  which  has  conformed 
to  all  the  conditions  necessary  to  make  it  the  cause  of  the 
disease. 

Origin. — In  the  tissues  and  all  the  body-fluids  and  secretions 
of  affected  individuals. 

Form. — Short,  thick  rods  with  an  indistinct  capsule,  rounded 
ends.  Growing  in  chains  in  fluid  media. 

Properties. — Immotile.  Stains  readily.  No  spores.  Culti- 
vated best  in  oxygen,  but  is  facultative  anaerobic.  Stains 
stronger  at  the  ends,  producing  bipolar  appearance.  Gelatin 
not  liquefied.  Easily  destroyed  by  sunlight  and  drying.  Very 
resistant  to  cold. 

Growth.— Best  at  37°  C. 

Gelatin. — At  22°  C.,  in  twenty-four  hours,  white,  point-like 
colonies  on  the  plates,  with  broad  and  flat  surface,  turning  gray 
and  then  brown. 

Stab. — Snow-white,  spreading  out  on  the  surface  to  the  edge, 
and  fluorescent. 

Bou'llon. — Granular  precipitate,  with  clear  fluid  above. 

AgLr  and  Blood-serum. — Glass-like  colonies  like  drops  of  dew 
at  first,  then  growing  larger  with  iridescent  edges. 

Potato. — At  37°  C.  small  white  mass. 

No  gas-jormation  in  glucose  media. 

Staining  readily  with  all  basic  dyes. 

Pathogenesis. — After  subcutaneous  injection  in  rats  death  fol- 
lows in  forty  to  sixty  hours,  with  symptoms  of  severe  toxemia 
and  convulsions.  The  point  of  infection  shows  a  local  edema 
and  inflammation  of  the  lymphatics.  All  the  organs  congested 
and  surrounded  by  a  bloody  exudate.  The  characteristic  bacilli 
in  all  the  tissues  and  secretions.  Nearly  all  the  domestic 
animals  are  susceptible.  Mosquitoes  and  pigeons,  however, 
are  immune — flies  are  not;  fleas  are  a  very  important  element 
in  the  transmission,  and  the  rat-flea  may  communicate  the 
disease  to  the  rat  from  man  or  from  the  rat  to  man.  Animals 
protected  from  the  flea  may  live  near  infected  animals  without 


PATHOGENIC    BACTERIA  179 

danger.  Direct  infection  by  dust  or  other  material  seldom 
occurs. 

Products. — A  toxin  has  been  obtained  and  immunity  has  been 
effected;  the  serum  of  immune  animals  has  protective  prop- 
erties. The  serum  likewise  shows  agglutinating  powers,  as 
with  typhoid  and  cholera  serums. 

Habitat. — Not  found  in  water,  but  most  likely  spreads  from 
the  soil  in  damp  and  darkened  areas.  Rats  become  affected 
first,  and  then  through  fleas  affect  man  and  other  animals. 
In  man  three  forms  of  the  disease  are  recognized  according  to 
the  mode  of  infection  and  course  of  the  disease — viz.,  bubonic, 
pulmonic,  septicemic. 

Vaccines. — The  vaccines  of  Haffkine  and  Terni  and  Bandi 
have  been  used  extensively,  and  with  some  good  results. 

Antitoxins. — The  antitoxins  of  Yersin  and  of  Lustig  have 
been  used,  but  without  much  result. 

Bacillus  Dysenteriae  (Shiga,  18p8). — The  term  dysentery 
is  applied  to  an  intestinal  disease  displaying  more  or  less  con- 
stancy in  its  clinical  manifestations,  but  having,  as  is  now  known, 
a  variety  of  causative  agents.  It  is  fairly  certain  that  one  type 
is  the  result  of  infection  with  an  ameba,  while  non-amebic 
forms  can  probably  be  produced  by  several  bacteria.  Chief 
among  those  is  the  bacillus  first  described  by  Shiga  in  Japan, 
and  since  then  found  by  Kruse  in  Germany,  by  Flexner,  Strong, 
and  Harvie  in  the  Philippine  Islands,  and  by  Vedder  and  Duval 
in  the  United  States.  Although  it  is  not  absolutely  proved 
that  it  is  the  cause  of  the  disease,  still  the  fact  that  it  is  constantly 
present  in  the  feces  in  one  type  of  dysentery,  that  such  cases 
give  a  positive  agglutination  reaction,  the  production  of  a 
curative  serum  by  the  immunization  of  animals  with  pure 
cultures,  and  the  results  on  experiment  animals,  leave  little 
doubt  as  to  the  specificity  of  the  organism. 

Origin. — The  dejecta  of  dysenteric  patients. 

Form. — A  plump  bacillus  with  rounded  ends,  resembling  the 
typhoid  and  colon  bacilli. 

Properties. — Motility  doubtful,  but  numerous  flagella  have 
been  demonstrated.  Does  not  form  spores. 


l8o         ESSENTIALS  OF  BACTERIOLOGY 

Staining. — Stains  readily,  negative  to  Gram;  facultative 
anaerobe. 

Growth. — Best  at  37°  C.  Killed  by  ten  minutes'  exposure 
to  55°  C. 

Gelatin. — A  white  line  of  growth  along  puncture;  super- 
ficial growth  slight. 

Bouillon. — Uniform  clouding.  Indol  usually  not  produced; 
milk  not  coagulated. 

Agar. — Resembles  typhoid  bacillus. 

Potato. — Thin  whitish  layer,  turning  light  brown. 

No  gas -formation  in  glucose  or  lactose  media. 

Patho genesis. — Mice  and  guinea-pigs  die  in  one  or  two  days 
after  intraperitoneal  inoculation.  Rabbits  usually  recover, 
though  lesions  analogous  to  those  of  human  dysentery  have 
been  produced.  Dogs  die  in  five  or  six  days,  with  well-marked 
diarrhea. 

Products. — The  patient's  blood-serum  agglutinates  the  ba- 
cillus in  cases  in  which  it  can  be  cultivated  from  the  stools. 
The  reaction  is  absent  from  other  cases.  Shiga  has  reduced 
the  mortality  from  34.7  to  9  per  cent,  by  means  of  a  serum 
obtained  from  immunized  horses,  but  in  more  extensive  tests 
the  antidysenteric  serum  proved  of  little  value. 

Habitat. — Found  in  the  stools  and  in  shreds  of  mucous  mem- 
brane from  the  intestinal  walls. 

Bacillus  Aerogenes  Capsulatus  (Welch,  18gl). — Origin. 
—The  intestine  of  man  and  animals,  soil,  sewage,  and  water. 

Form. — A  thick  bacillus,  3  to  6  //  in  length,  frequently  capsu- 
lated. 

Properties. — Not  motile,  anaerobic,  forms  spores  chiefly  in 
cultures  on  blood-serum. 

Growth.— Best  at  37°  C. 

Gelatin. — Liquefied  slowly  or  not  at  all. 

Bouillon. — Forms  gas. 

Milk. — Coagulated  and  becomes  acid. 

Potato. — Thin,   grayish-white   growth   with   gas-production. 

Forms  gas  in  abundance  on  dextrose,  lactose,  or  saccharose 
media. 


PATHOGENIC   BACTERIA  l8l 

Pailio  genesis. — Is  not  usually  pathogenic  for  rabbits  and 
mice,  though  in  guinea-pigs  and  birds  it  produces  "gas  phleg- 
mons." It  is  sometimes  found  in  autopsies  on  human  subjects, 
producing  bubbles  or  cavities  in  the  viscera  (Schaumorgane) , 
but  this  is  probably  due  to  postmortem  migration  of  the  germ 
from  the  intestine.  It  has  been  recovered  from  the  blood  during 
life,  however,  and  is  the  most  frequent  cause  of  emphysematous 
gangrene.  Various  foreign  observers  have  described  organisms 
having  similar  properties,  and  have  given  them  such  names  as 


Fig.  in. — Bacillus  aerogenes  capsulatus  (from  photograph  by  Professor  Simon 

Flexner). 


Bacillus  perfringens,  Bacillus  enteritidis,  Granulobacillus 
immobilis,  etc.,  but  they  were  probably  dealing  with  the 
Bacillus  aerogenes  capsulatus. 

Micrococcus  Melitensis  (Bruce,  1887). — Malta  fever, 
also  known  as  Mediterranean  fever,  occurs  in  the  region  from 
which  it  derives  its  name,  but  has  been  observed  in  India,  the 
Philippine  Islands,  and  Porto  Rico.  Bruce  cultivated  a  micro- 
coccus  from  the  spleen  and  proved  its  specificity. 

Origin. — Is  found  most  abundantly  in  the  spleen. 


1 82  ESSENTIALS    OF    BACTERIOLOGY 

Form. — Rounded  or  oval,  5  p.  in  diameter,  singly,  in  pairs,  or 
short  chains. 

Properties. — Non-motile,  though  flagella'  said  to  be  present; 
grows  slowly,  best  at  body-temperature. 

Gelatin. — Not  liquefied;  growth  very  slow. 

Bouillon. — Turbid,  with  sediment. 

Agar. — Pearly  white  growths. 

Potato. — Slight  invisible  growth. 

Stained  by  ordinary  anilin  dyes. 

The  disease  may  be  produced  in  monkeys  by  even  small 
amounts  of  pure  culture.  In  man  a  chronic,  remittent  febrile 
disease  is  produced,  with  sweating  and  arthritis.  The  mortality 
is  2  per  cent.  A  serum  reaction  can  be  obtained  and  is  diag- 
nostic. 

Microorganisms  have  been  found  by  various  observers  in 
measles,  scarlatina,  mumps,  and  whooping-cough,  but  their 
specificity  is  still  in  doubt. 

Mode  of  Transmission. — Zammitt  found  that  50  per  cent, 
of  the  goats  of  Malta  gave  the  agglutination  reaction  to  the 
micrococcus,  and  it  was  present  in  the  milk  in  10  per  cent. 
Monkeys  fed  on  the  milk  contracted  the  disease. 

Preventive  measures  instituted  in  1906  have  borne  out  the 
theory  that  the  milk  of  goats  is  the  cause  of  Malta  fever,  and 
since  the  practice  of  importing  goats  from  Malta  has  stopped, 
the  disease  has  disappeared  from  Gibraltar.  In  Malta,  among 
the  troops,  the  fever  has  been  greatly  reduced  by  eliminating 
milk  from  the  dietary. 

Bacillus  Enteritidis  Sporogenes  (Klein) . — Origin. — First 
isolated  from  stools  of  infantile  diarrhea.  It  is  found  in  sewage. 

Form. — Bacillus  twice  as  long  as  it  is  broad,  often  containing 
a  spore  at  one  end.  Is  slightly  motile  and  has  flagella. 

Growth. — Grows  well  under  anaerobic  conditions  in  ordinary 
media.  Liquefies  gelatin  in  twenty-four  hours,  produces 
acid  and  gas  in  bile-salt  glucose  media.  In  milk  it  separates 
the  curd  in  twenty-four  hours,  with  abundant  gas-formation. 

Pathogenesis. — If  a  small  quantity  of  the  milk  culture  is 
inoculated  into  a  guinea-pig,  the  animal  dies  in  twenty-four 


PROTOZOA  183 

hours.  The  skin  around  the  point  of  inoculation  becomes 
gangrenous,  and  foul-smelling  edema,  with  gas-containing 
blebs,  occurs. 

Habitat. — It  is  a  common  inhabitant  of  the  intestines  of 
man  and  animals,  and  if  found  in  water,  is  a  sure  indication 
of  sewage  pollution. 


CHAPTER  XX 
PROTOZOA 


PROTOZOA  are  unicellular  animal  organisms,  minute  as  bac- 
teria, and  differing  from  bacteria  in  the  methods  of  repro- 
duction. Their  structure  and  functions  are  more  complex, 
although  the  borderland  is  ill  denned. 


Fig.  112. — Pure  culture  of  trypanosomes  of  mosquitoes — Crithidia  fas- 
ciculata.  Multiplication  roset  showing  large  and  small  cells.  Nine-day 
culture  (Gen.  i.  x  1500)  (Novy,  MacNeal,  and  Torrey). 

Divisions. — There  are  four  grand  divisions  of  protozoa: 
(i)  Sarcodina,  containing  5500  species;  (2)  mastigophora, 


184  ESSENTIALS    OF    BACTERIOLOGY 

containing  500  species;  (3)  infusoria,  containing  700  species; 
(4)  sporozoa,  containing  300  species. 

Sarcodina  are  chiefly  marine  forms,  with  processes  change- 
able in  shape. 

Mastigophora  have  undulating  flagella,  and  are  known  as 
flagellates;  to  this  division  the  trypanosomata  belong. 

Infusoria  have  fine  ciliary  processes  or  numerous  delicate 
flagella. 

Sporozoa  have  no  motile  organs,  and  are  reproduced  by  spores. 
To  this  division  belong  the  coccidia  of  malaria  and  the  organ- 
isms discovered  by  Mallory  in  scarlatina. 


Fig.  113. — Pure  culture  of  trypanosomes  of  mosquitoes — Crithidia  fas- 
ciculata.  Part  of  roset  of  elongated  crithidia  with  flagella  directed  centrally 
(Gen.  39,  Xisoo)  (Novy,  MacNeal,  and  Torrey). 

Life-cycle. — The  complete  cycle  of  reproduction  has  been 
observed  in  only  one  of  the  pathogenic  protozoa,  namely,  the 
protozoa  of  malaria. 

Life  Cycle  of  the  Malarial  Sporozoa. — According  to  its 
situation,  the  parasite  exhibits  two  distinct  phases  of  existence: 
in  the  human  blood  it  passes  through  an  asexual  reproductive 
cycle,  known  as  schizogony,  while  in  the  body  of  the  mosquito 
it  undergoes  an  entirely  different  series  of  sexually  reproductive 
changes,  called  sporogony.  It  is  simpler  first  to  describe  the 


PROTOZOA  185 

life  history  of  the  organism  in  general,  pointing  out  the  differ- 
ences shown  by  the  varieties  later. 

i.  The  Asexual  Cycle  in  Man. — An  infected  mosquito  con- 
veys the  parasites  into  the  blood  as  minute  hyaline  bodies 
which  enter  the  blood-cells.  At  first  they  are  small,  round, 
colorless  bodies,  exhibiting  more  or  less  active  ameboid  motion 
in  the  fresh  blood.  Sometimes,  particularly  in  the  estivo- 
autumnal  form,  a  ring  shape  is  assumed.  Their  size  gradually 
increases  and  pigment-granules  appear,  while  in  stained  speci- 
mens a  nucleus  containing  chromatin  granules  is  visible.  As 


Fig.  114. — Pure  culture  of  trypanosomes  of  mosquitoes — Crithidia  fas- 
ciculata.  Elongated  crithidia  from  same  preparation  as  preceding  (Novy, 
MacNeal,  and  Torrey). 

the  parasite  approaches  maturity,  the  chromatin  becomes 
scattered,  and  finally  the  protoplasm  or  mother-cell,  known  as 
sporocyte,  divides  into  six  to  twenty  spores,  daughter-cells  or 
merozoites,  each  containing  a  portion  of  the  chromatin.  The 
number  of  spores  formed  and  their  arrangement  before  seg- 
mentation takes  place  differ  in  the  three  varieties  and  will  be 
noted  below.  The  spores  burst  through  the  envelop  of  the  red 
corpuscle  and  become  free  in  the  blood,  but  speedily  enter 
fresh  corpuscles  and  pass  through  the  same  series  of  changes. 
The  febrile  stage  is  synchronous  with  sporulation  and  liberation 
of  the  young  forms. 


i86 


ESSENTIALS    OF    BACTERIOLOGY 


Certain  of  the  parasites  do  not,  however,  go  on  to  segmenta- 
tion, but,  after  reaching  maturity,  remain  quiescent  and  form 
the  so-called  gametes  or  sexual  types.  In  the  tertian  and 


/    i     V3        TH«  EMDOCiCMCrUS  OR 
(/\  ft0£    ASEXUAL,  CYCLE. 

11  0 


THE 

MOSQUITO  PHASE: 
EXOGENOUS 


•^SEXUAL  CVCLE-. 


Fig.  115. — Schema  showing  the  human  and  mosquito  cycles  of  the  malarial 
parasite:  A,  Normal  red  cell;  B,  C,  D,  E,  red  cells  containing  amebulas  or 
myxopods;  F,  G,  H,  sporocytes;  J',  K',  L',  M',  microgametocytes  or  male 
gametes;  J",  K",  L",  M",  O,  macrogametocytes,  or  female  gametes;  N',  N', 
microgametes;  P,  traveling  vermicule;  Q,  young  zygote;  R,  S,  zygotomeres; 
T,  blastophore;  U,  mature  zygote  (modified  from  Blanchard's  diagram  illus- 
trating life-cycle  of  Coccidium  schubergi)  (Rees,  in  "Practitioner,"  March, 
1901). 

quartan  varieties  these  are  not  very  different  from  the  mature 
organisms,  but  the  estivo-autumnal  gametes  are  crescentic  in 
shape  and  very  characteristic. 


PROTOZOA  187 

2.  The  Sexual  Cycle  in  the  Mosquito. — If,  now,  the  blood  is 
shed,  certain  of  the  gametes  (the  male  forms  or  microgameto- 
cytes)  extrude  long  protoplasmic  processes  containing  a  central 
core  of  chromatin,  and  which  represent  the  male  fertilizing 
element  (micro gametes] .  These  become  detached,  and,  entering 
a  female  gamete  (macrogamete) ,  a  true  sexual  fertilizing  process 
takes  place.  In  the  alimentary  canal  of  the  mosquito  these 
fertilized  cells  penetrate  the  stomach-walls  and  form  cysts 
(oocysts)  filled  with  a  large  number  of  filiform  spores  (sporo- 
zoites),  which  are  extruded  into  the  body  cavity  of  the  insect, 
and  some  of  which  reach  the  salivary  glands,  whence  they  are 
ejected  when  the  mosquito  bites.  This  cycle  of  development 
takes  seven  or  eight  days. 

Three  Forms  of  Malarial  Protozoa.— i.  The  Tertian 
Form. — The  adult  forms  are  large,  not  very  refractile,  and 
their  outline  is  somewhat  indistinct.  There  is  an  abundance 
of  fine  pigment-granules,  and  the  ameboid  motion  is  vigorous. 
Segmenting  forms  divide  into  fifteen  to  twenty  merozoites;  the 
sexual  forms  or  gametes  are  large.  The  red  cell  containing  the 
organism  is  swollen  and  pale.  Sporulation  and,  therefore,  the 
malarial  paroxysm  occur  every  forty-eight  hours. 

2.  The  Quartan  Form. — The  organism  is  smaller,  is  more 
refractile,  and  its  outline  is  more  distinct.     The  pigment  is 
coarse  and  situated  at  the  periphery  of  the  organism,  while  the 
protoplasmic   motion   is   sluggish.     Segmentation   forms   only 
six  to  twelve  spores,  and  has  the  regular  "daisy-head"  appear- 
ance; the  gametes  are  small.     The  red  cells  become  dark  in 
color,  and  the  cycle  requires  seventy-two  hours. 

3.  Estivo -autumnal    Form. — The    adult    forms    are    found 
mainly  in  the  spleen  and  other  viscera,  and  do  not  very  often 
occur  in  the  peripheral  blood;  their  outline  is  sharp,  and  they 
are  highly  refractile.     The  pigment  is  scanty  and  fine;  the 
motion  is  active.     A  variable  number  of  merozoites  is  formed — 
usually  six  to  twelve.     The  gametes  are  characteristic,  being 
crescentic  in  shape  and  very  resistant  to  quinin.     The  red  cell 
becomes  shriveled  and  yellowish.      The  cycle  usually  takes 
forty-eight  hours,  though  it  is  somewhat  variable. 


i88 
/ 


ESSENTIALS    OF    BACTERIOLOGY 


J2 


10 


! 


o 


13 


15 


J8 


13 


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J6 

> 


20 


22 


c  •      h 

t          ««•   -1 

c-  - .- J 


27 


Fig.  116. 


PROTOZOA  189 

Mixed  infections  with  the  different  organisms  or  with  two  or 
more  broods  of  the  same  organism  may  occur,  so  that  quotidian 
and  irregular  paroxysms  may  be  produced. 

Transmission. — Malaria  is  spread  by  means  of  a  mosquito, 
the  anopheles,  in  whose  body  the  protozoon  undergoes  its 
highest  development.  Man  is  the  intermediate  host. 

Methods  of  Examination  for  Malarial  Organisms. — 
i .  Fresh  preparations  are  made  by  placing  a  small  drop  of  blood 
on  a  slide  and  a  cover-glass  over  it,  so  that  only  a  thin  film  is 
formed.  A  ring  of  vaselin  is  smeared  over  the  edges  of  the 
cover-glass  to  prevent  evaporation.  This  is  the  best  method 
for  studying  flagellation  and  fertilization,  but  is  less  satisfactory 
for  routine  clinical  work  than — 

2.  Stained  Smears. — These  are  made  by  spreading  a  drop  of 
blood  in  a  thin  film  over  one  slide  with  the  edge  of  another, 
drying  in  the  air,  and  staining.  Many  stains  have  been  devised 
for  the  malarial  organism,  but  the  following  are  sufficient  for 
ordinary  use: 

(1)  Marchoux's  Thionin  Stain. — Add  20  c.c.  of  saturated 
solution  of  thionin  in  50  per  cent,  alcohol  to  100  c.c.  of  2  per 
cent,  phenol.     Fix  the  smears  and  stain  for  fifteen  to  twenty 
.seconds.     The  malarial  organisms  are  stained  a  deep  purple, 
strongly  contrasting  with  the  faint  green  of  the  red  cells,  so 
that  they  are  readily  recognized. 

(2)  Jenner's  Stain. — This  is  excellent  for  routine  work,  as  no 
preparatory  fixation  is  required.     Equal  parts  of  a  1.2  per  cent, 
aqueous  solution  of  Griibler's  water-soluble  eosin  and  a  i  per 
cent,  aqueous  solution  of  Grlibler's  medicinal  methylene-blue 
are  mixed,  and  the  resulting  precipitate  allowed  to  stand  for 
twenty-four  hours,  washed,  and  dried.     Half  a  gram  of  this  is 

Fig.  116. — Various  forms  of  malarial  parasites  (Thayer  and  Hewetson): 
i-io  Inclusive,  tertian  organisms;  11-17  .inclusive,  quartan  organisms;  18-27 
inclusive,  estivo-autumnal  organisms. 

i,  Young  hyaline  form;  2,  hyaline  form  with  beginning  pigmentation; 
3,  pigmented  form;  4,  full-grown  pigmented  form;  5,  6,  7,  8,  segmenting  forms; 
9,  mature  pigmented  form;  10,  flagellate  form. 

n,  Young  hyaline  form;  12,  13,  pigmented  forms;  14,  fully  developed 
form;  15,  16,  segmenting  forms;  17,  flagellate  form. 

18,  19,  20,  Ring-like  and  cross-like  hyaline  forms;  21,  22,  pigmented 
forms;  23,  24,  segmenting  forms;  25,  26,  27,  crescents. 


ESSENTIALS    OF    BACTERIOLOGY 

dissolved  in  100  c.c.  of  pure  methyl-alcohol.  The  smears  are 
dropped  into  this  stain  for  one  to  three  minutes,  without  pre- 
vious fixation,  and  at  once  rinsed  in  distilled  water.  The 
malarial  parasites  are  stained  blue,  the  cell-bodies  a  reddish 
brown. 

(3)  Wright's  Chromatin  Stain.— This  is  the  best  of  the 
chromatin  stains.  For  its  preparation,  which  is  quite  com- 
plicated, see  Wright,  Journal  of  Medical  Research,  vol.  vii,  1902. 
It  is  used  as  follows: 

1.  The  stain  is  poured  over  the  film  and  allowed  to  remain 
for  one  minute  to  secure  fixation. 

2.  Add  distilled  water  drop  by  drop  until  a  metallic  scum  is 
formed  on  the  surface.     The  staining  now  takes  place  and 
requires  two  to  three  minutes.     Wash  in  distilled  water  until  a 
pinkish  tint  appears  in  the  thin  portions  of  the  smear.     The 
body  of  the  malarial  parasite  is  stained  blue,  and  its  chromatin 
a  lilac  to  red  color.     The  red  cells  are  orange  pink. 

If  possible,  examinations  for  malarial  organisms  should 
always  be  made  before  quinin  is  administered. 

Trypanosomata. — Trypanosomes  are  flagellate  protozoa 
found  in  the  blood  of  various  animals,  and  causing  a  number  of 
diseases,  such  as  surra,  dourine,  and  nagana,  affecting  horses 
and  cattle,  especially  in  tropical  countries,  and  causing  the 
sleeping  sickness  of  Africa,  which  is  very  fatal  for  human  beings. 

Morphology, — A  fusiform  mass,  containing  at  one  end  a 
flagellum. 

In  the  living  state  these  protozoa  are  very  motile.  In  the 
stained  specimen  chromatin  granules  are  found  and  two  or 
more  nuclei.  From  the  smaller  nucleus  arises  the  undulatory 
membrane,  which  passes  into  the  flagellum  and  assists  in  the 
wave-like  motion. 

In  the  body  fluids  division  occurs,  first  of  the  nucleus,  and 
then  of  the  protoplasm. 

Cultivation. — Novy  and  MacNeal  have  succeeded  in  culti- 
vating these  protozoa  on  blood-agar,  and  multiplication  goes 
on  rapidly,  so  that  rosets  are  formed  with  the  flagella  arranged 
around  a  common  center.  (See  Figs.  112,  113,  114.) 


PROTOZOA  IQI 

Trypanosoma  Lewis!  (Lewis,  1878). — Found  in  rats;  not 
fatal  to  them,  though  often  equaling  the  red  corpuscles  in 
number.  The  infection  continues  for  two  months  without  pro- 
ducing any  illness,  and  the  animal  is  then  immune. 

Cultivated  best  at  20°  C.  and  very  resistant  to  cold.  The 
rat  is  probably  infected  by  the  bite  of  a  flea  or  louse.  (See  Fig. 
117.) 

Trypanosoma  Brucei  (Bruce,  1894)  causes  nagana,  or 
tsetse-fly  disease,  a  disease  affecting  horses,  cattle,  and  dogs 
in  certain  regions  of  South  Africa.  The  trypanosome  of  Bruce 


Fig.  117. — Trypanosome  from  blood  of  gray  rat;  stained  with  a  2  per  cent, 
aqueous  solution  of  methylene-blue  (Boston). 

is  less  motile  than  that  of  Lewis.  It  has  been  cultivated  at 
25°  C.,  and  is  less  resistant  to  cold.  All  laboratory  animals 
subject  to  infection.  The  rat  dies  in  ten  days. 

In  the  natural  infection  Bruce  discovered  that  the  tsetse-fly 
transmitted  the  disease,  but  that  it  did  so  by  first  biting  some 
animal  whose  blood  contained  the  trypanosome.  The  blood 
of  infected  animals  contains  the  organism,  and  can,  if  injected, 
produce  the  disease  without  the  agency  of  the  fly.  So  far  the 
tsetse  fly  alone  is  responsible  for  the  spread  of  the  infection. 


IQ2  ESSENTIALS    OF    BACTERIOLOGY 

Trypanosoma  Ugandense  Gambiense  (T.  Castellani, 
T.  Hominis,  T.  Neprevi). — Sleeping  sickness,  or  human  try- 
panosomiasis,  is  a  disease  peculiar  to  some  parts  of  Africa.  It 
is  accompanied  by  periods  of  fever,  anemia,  and,  finally,  a 
lethargy  deepening  into  coma  and  death.  The  disease  may 
be  rapid,  and  it  may  last  with  recurrences  for  many  years. 
Trypanosomes  identical  with  those  found  in  nagana  disease 
have  been  found  in  the  blood  of  infected  persons,  and  described 
by  various  observers,  and  given  different  names. 

Monkeys,  when  inoculated  with  cerebrospinal  fluid  from 
affected  persons,  develop  a  similar  disease,  and  the  parasites 
are  found  in  the  blood. 

A  blood-sucking  fly,  known  as  the  Glossina  palpalis,  is  con- 
sidered the  means  of  infection.  The  fly  is  closely  related  to  the 
Glossina  morsitans,  or  tsetse  fly.  The  sleeping  sickness  in 
man  is  most  likely  the  same  thing  as  the  nagana  of  cattle. 

Trypanosoma  Evansi  (Evans,  1880). — Pathogenic  for 
all  animals. 

Discovered  in  the  blood  of  horses  suffering  from  surra,  a 
disease  prevalent  in  India  and  the  Philippine  Islands.  The 
disease  resembles  nagana. 

T.  equiperdum  and  T.  Rougetii  are  names  given  to  similar 
organisms  found  in  dourine,  a  disease  affecting  horses  in 
southern  France  and  Spain.  Trypanosomes  are  found  in 
fish,  oysters,  birds,  and  frogs,  and  many  varieties  have  been 
described. 

Piroplasma  Bovis  (P.  Bigeminum)  (T.  H.  Smith,  1893). 
— Origin. — In  the  blood  of  animals  suffering  from  Texas  cattle- 
fever. 

Form. — A  pear-shaped  protozoon,  found  in  pairs  in  the  red 
cells  of  the  blood,  the  smaller  ends  of  pear  in  opposition;  coarse 
ameboid  movement. 

Transmission. — An  insect  or  tick  (Boophilis  bovis)  becomes 
infected,  and  by  its  bite  infects  other  animals. 

Other  similar  sporozoa  have  been  found  in  animal  diseases 
and  in  man  in  Rocky  mountain  fever.  The  P.  hominis  has 
been  described,  but  not  definitely  determined. 


PROTOZOA  193 

Negri  Bodies  (Negri,  1903). — Origin. — Found  in  the 
nervous  system  of  animals  dying  of  rabies  (hydrophobia) . 

Form. — Round  and  oval,  hyaline  bodies,  with  a  sharp  outline 
and  containing  a  nucleolus.  The  plasma  is  slightly  granular. 
They  are  regarded  as  protozoa. 

Staining. — A  smear  from  brain  tissue  is  made  on  a  cover- 
glass  and  fixed  in  methyl-alcohol  for  five  minutes;  then  stained 
by  Giemsa;  stain  for  half -hour  to  three  hours. 

Spirillum  of  Relapsing  Fever  (Obermeier,  1873).— Syn- 
onym.— Spirochaeta  Obermeieri. 


Fig.  118. — Spirochaeta  Obermeieri  from  human  blood  (Kolle  and  Wasser- 

mann). 

The  definite  classification  of  this  organism  has  not  been 
made.  Some  regard  it  now  as  a  protozoon,  and  one  of  a  group 
in  which  numerous  other  spirilla  belong. 

Origin. — Found  in  the  blood  of  recurrent  fever  patients, 
described  in  1873. 

Form. — Long,  wavy  threads  (16  to  40  //  long),  a  true  spiril- 
lum; flagella  are  present  (Fig.  118). 

Properties. — Very  motile.     Has  not  been  cultivated. 

Staining. — Ordinary  anilin  stains.  Bismarck-brown  best 
for  tissue  sections. 

Pathogenesis. — Found  in  the  organs  and  blood  of  recurrent 
fever.  Man  and  monkeys  inoculated  with  blood  from  one  suf- 
13 


IQ4  ESSENTIALS   OF   BACTERIOLOGY 

fering  from  this  disease  become  attacked  with  the  fever,  and 
in  their  blood  the  spirillum  is  again  found.  It  is  found  in  the 
blood,  only  in  the  relapses  (during  the  fever) .  After  the  attack 
the  spirilla  gather  in  the  spleen  and  gradually  die  there.  It 
has  been  found  in  the  brain,  spleen,  liver,  and  kidneys.  In  the 
secretions  it  has  not  been  discovered. 

Agglutinating  substances  have  been  developed.  Immunity 
has  been  produced  in  rats,  and  the  serum  has  antitoxic  proper- 
ties. 

Transmission. — The  bed-bug  retains  the  spirillum  in  its 
blood  and  is  considered  an  important  factor  in  spreading  the 
disease. 

African  Tick  Fever. — A  spirochaete  similar  to  that  of  relapsing 
fever  has  been  observed  in  ticks,  which  conveyed  a  disease  to 
monkeys  similar  to  the  above  fever. 

Spirochaete  Pallida  (Schaudinn,  IpOs) ;  Spironema 
Pallidum;  Treponema  Pallidum. — Found  in  hereditary 


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-  .•  \  %'     w^.**'-'   '% 


^(B    - 

fc 

^   ^*w 


Fig.  119. — Spirochaeta  pallida.     Microphotograph  made  by  Dr.  R.  E.  Laven- 
son  from  a  specimen  prepared  by  H.  Fox  (Stengel). 

syphilis  in  all  organs,  in  chancre,  and  lymphatic  glands,  and 
in  secondary  lesions. 

Form. — A  minute,  spiral-shaped  organism,  with  six  to  eight 
curves,  ends  tapering.  Actively  motile  in  fresh  specimen  (Fig, 
119). 


PROTOZOA  195 

Staining. — The  organism  requires  special  staining,  and  a 
number  of  complicated  methods  have  been  introduced  by 
different  investigators.  The  Giemsa  stain  is  said  to  give  the 
best  results.  (See  Staining  Fluids,  p.  35-) 

The  slide  is  fixed,  dried  in  air,  hardened  in  absolute  alcohol 
twenty-five  minutes,  stained  with  dilute  stain  (i  drop  to  i  c.c. 
of  water)  for  ten  minutes,  washed  in  water,  and  mounted. 

In  tissues  the  organism  can  be  shown  by  fixing  with  silver 
nitrate  after  the  manner  of  Ramon  y  Cajal.  The  tissue  is — 
(i)  Hardened  in  formalin  for  twenty-four  hours  (the  sections 
should  be  thin) ;  (2)  washed  in  water  for  one  hour;  (3)  alcohol, 
twenty-four  hours;  (4)  i^  per  cent,  silver  nitrate  solution  in 
incubator  at  37°  C.  three  days;  (5)  washed  in  water  twenty 
minutes;  (6)  placed  in  mixture  of  pyrogallic  acid,  4  parts;  for- 
malin, 5  parts;  distilled  water,  to  make  100  parts,  and  kept  in 
dark  bottle  for  forty-eight  hours;  (7)  washed  in  water  and 
alcohol  and  then  embedded  in  paraffin  and  sectioned.  Spiro- 
chaetae  black,  tissues,  pale  yellow.  Or  counterstain  of  fuchsin 
can  be  employed. 

Transmission. — Metchnikoff  and  Roux  have  inoculated 
anthropoid  apes,  producing  in  them  the  primary  sore  and 
secondary  symptoms,  with  reproduction  of  the  organism. 
There  is  no  increase  in  the  blood.  All  facts  point  to  this 
organism  as  being  the  true  cause  of  syphilis. 

Classification. — It  is  undecided  whether  the  spirochaete  of 
syphilis  belongs  to  the  group  of  bacteria  known  as  spirilla  or 
to  the  protozoa. 

Amoeba  Dysenteriae. — Found  in  the  intestinal  ulcers,  feces, 
and  secondary  liver  abscesses  in  certain  cases  of  dysentery.  A 
non-pathogenic  form,  Amoeba  coli,  also  occurs.  The  Amoeba 
dysenteriae  is  a  unicellular  animal  organism,  measuring  25  to 
35  fj.  in  diameter,  though  larger  and  smaller  forms  occur.  There 
are  a  nucleus  and  a  nucleolus;  the  protoplasm  of  the  cell-body 
is  vacuolated,  and  often  contains  red  blood-cells  and  bacteria. 
In  fresh,  warm  stools  active  ameboid  motion  may  be  observed. 
The  non-pathogenic  form  is  smaller  and  never  contains  red 
blood-cells. 


196  ESSENTIALS  OF  BACTERIOLOGY 

Small-pox  and  Vaccinia. — The  exciting  agent  of  small-pox 
is  still  unknown,  but  numerous  bacteria  and  protozoon-like 
bodies  have  been  described  and  given  etiologic  significance  by 
various  authors.  There  is  some  evidence  in  favor  of  Funck's 
belief  that  vaccinia  is  caused  by  a  protozoon,  the  Sporidium 
vaccinale.  Animals  inoculated  with  this  organism  developed 
both  vaccinia  and  variola. 

Yellow  Fever. — For  some  years  it  was  thought  that  a  bacil- 
lus, called  Bacillus  icteroides  by  Sanarelli,  was  the  cause  of 
yellow  fever.  The  earlier  work  of  Sternberg  was  disproved 
when  it  was  shown  that  his  bacillus,  Bacillus  X,  was  identical 
with  the  colon  group,  and  Reed  and  Carroll  found  that  San- 
arelli's  germ  was  an  allied  organism. 

It  is  now  known  that  a  special  species  of  mosquito,  Stegomyia 
fasciata,  conveys  the  infection  and  acts  as  a  culture-medium  for 
some  unknown  microorganism,  possibly  a  protozoon,  which 
must  undergo  certain  changes  to  become  virulent. 

Only  by  the  bite  of  a  mosquito  infected  with  the  blood  of  a 
yellow-fever  patient  or  by  direct  inoculation  of  such  blood  can 
yellow-fever  be  transmitted. 

The  experiments  made  so  far  show  that  the  germ  is  destroyed 
by  a  temperature  of  55°  C.  for  ten  minutes.  It  can  pass 
through  a  Berkefeld  filter,  and  is,  therefore,  extremely  minute, 
but  no  one  has  as  yet  been  able  to  find  any  distinctive  organism 
in  any  of  the  blood. 


CHAPTER  XXI 

BACTERIA  PATHOGENIC  FOR  ANIMALS  BUT  NOT  FOR 

MAN 

A  GREAT  many  bacteria  have  been  described  in  diseases 
peculiar  to  the  lower  animals  which  have  only  slight  patho- 
genicity  for  man,  and  only  some  of  the  more  prominent 
varieties  can  be  treated  of  here. 

Bacillus  of  Symptomatic  Anthrax  (Bellinger  and  Feser) . 


BACTERIA   PATHOGENIC    FOR   ANIMALS,    NOT    FOR  MAN   Ip7 

— Synonym. — Charbon  symptomatique,  Arloing,  Cornevin,  and 
Thomas. 

Origin. — This  bacillus,  described  already  in  1879,  has  been 
isolated,  and  by  animal  inoculation  shown  to  be  the  cause  of 
the  "black-leg"  or  " quarter-evil "  disease  of  cattle. 

Form. — Large  slender  rods,  which  swell  up  at  one  end  or  in 
the  middle  for  the  spore  (Fig.  120). 

Properties. — They  are  motile,  and  liquefy  gelatin  quite 
rapidly. 


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Fig.  120. — Bacilli  of  symptomatic  anthrax,  with  spores  (x  1000)  (Frankel 
and  Pfeiffer). 

A  rancid  odor  is  developed  in  the  cultures. 

Cultures. — The  growth  occurs  slowly,  and  only  in  an  atmo- 
sphere of  hydrogen,  being  very  easily  destroyed  by  oxygen 
and  carbon  dioxid;  grows  best  at  38°  C.;  under  15°  C.  no 
growth. 

Glucose-gelatin. — In  a  few  days  little  round  colonies  develop, 
which,  under  low  power,  show  hairy  processes  around  a  compact 
center. 

Stab-cultures  in  Full  Test-tubes. — The  first  growth  in  the 
lower  portion  of  the  tube  not  very  characteristic.  Gases 
develop  after  a  few  days,  and  the  gelatin  becomes  liquid. 


198  ESSENTIALS  OF  BACTERIOLOGY 

Agar  at  brood  temperature,  in  twenty-four  to  forty-eight 
hours,  an  abundant  growth  with  a  sour  odor  and  abundant 
gas-formation. 

Staining. — Ordinary  methods.  Gram's  method  is  negative, 
but  the  spores  can  be  colored  by  the  regular  double  stain  for 
spores. 

Pathogenesis. — If  a  small  amount  of  the  culture  be  injected 
under  the  skin  of  a  guinea-pig,  in  twenty  hours  a  rise  of  tempera- 
ture, pain  at  the  site  of  injection,  and  in  a  few  hours  more 
death  occurs.  At  the  autopsy,  the  tissues  are  found,  blackened 
in  color  and  soaked  with  a  bloody  serous  fluid;  in  the  connective 
tissue  large  collections  of  gas,  but  only  in  the  neighborhood  of 
the  point  of  infection.  The  bacilli  are  found  in  great  num- 
bers in  the  serum,  but  only  appear  in  the  viscera  some  time 
after  death,  when  spores  have  developed. 

The  animals  are  usually  infected  through  wounds  on  the 
extremities;  the  stalls  or  meadows  having  been  soiled  by  the 
spore-containing  blood  of  animals  previously  dead  of  the  dis- 
ease. " Rautehbrand"  is  the  German  name;  "Charbon  symp- 
tomatique,"  the  French,  from  the  resemblance  in  its  symptoms 
to  anthrax. 

Immunity. — Rabbits,  dogs,  pigs,  and  fowl  are  immune  by 
nature,  but  if  the  bacilli  are  placed  in  a  20  per  cent,  solution  of 
lactic  acid  and  the  mixture  injected,  the  disease  develops  in 
them.  The  lactic  acid  is  supposed  to  destroy  some  of  the 
natural  resistance  of  the  animal's  cells. 

When  a  bouillon  culture  is  allowed  to  stand  a  few  days, 
the  bacilli  therein  lose  their  virulence,  and  animals  are 
no  longer  affected  by  them;  but  if  they  are  placed  in  20 
per  cent,  lactic  acid  and  the  mixture  injected,  their  virulence 
returns. 

Immunity  is  produced  by  the  injections  of  these  weakened 
cultures,  and  also  by  some  of  the  products  which  have  been 
obtained  from  the  cultures. 

Bacillus  of  Chicken  Cholera  (Pasteur). — Synonyms  — 
Micrococcus  cholera  gallinarum;  Microbe  en  huit;  Bacillus 
amcidus;  bacillus  of  fowl  septicemia. 


BACTERIA   PATHOGENIC    FOR   ANIMALS,    NOT   FOR   MAN   199 

Origin. — In  1879  Perroncito  observed  this  cocci-like  ba- 
cillus in  diseases  of  chickens,  and  Pasteur,  in  1880,  isolated 
and  reproduced  the  disease  with  the  bacillus  in  question. 

Form. — At  first  it  was  thought  to  be  a  micro- 
coccus,  but  it  has  been  found  to  be  a  short  rod, 
about  twice  as  long  as  it  is  broad,  the  ends 
slightly  rounded.  The  center  is  very  slightly 
influenced  by  the  anilin  colors,  the  poles 
easily,  so  that  in  stained  specimens  the  bacil- 
lus looks  like  a  dumb-bell  or  a  figure-of-eight  chickl'n 
(Microbe  en  huit) .  in  blood  (x  1000) 

T-,  , .  mi          i  i  f  (Frankel        and 

Properties. — They  do  not  possess  self-move-     pfeiffer). 

ment;  do  not  liquefy  gelatin. 

Growth. — Occurs  at  ordinary  temperature,  requiring  oxygen 
for  development.  It  grows  very  slowly. 

Gelatin  Plates. — In  the  course  of  three  days  little  round, 
white  colonies,  which  seldom  increase  in  size,  having  a  rough 
border  and  very  finely  granulated. 

Stab-cultures. — A  very  delicate  gray  line  along  the  needle- 
track,  which  does  not  become  much  larger. 

A  gar  Stroke  Culture. — A  moist,  grayish-colored  skin,  more 
appreciable  at  brood-heat. 

Potato. — At  brood-heat,  after  several  days,  a  very  thin,  trans- 
parent growth. 

Staining. — Methylene-blue  gives  the  best  picture.  Gram's 
method  is  not  applicable.  As  the  bacillus  is  easily  decolorized, 
anilin-oil  is  used  for  dehydrating  tissue  sections,  instead  of 
alcohol. 

Method: 

Loffler's  methylene-blue J  hour 

Alcohol 5  seconds 

Anilin-oil 5  minutes 

Turpentine i  minute 

Xylol  and  Canada  balsam. 

Pathogenesis. — Feeding  the  fowls  with  the  bacilli  or  inject- 
ing them  under  the  skin  will  cause  death  in  from  twelve  to 


200  ESSENTIALS   OF   BACTERIOLOGY 

twenty-four  hours,  the  symptoms  preceding  death  being  those 
of  a  severe  septicemia. 

The  bacillus  is  then  found  in  the  blood  and  viscera  and  the 
intestinal  discharges,  the  intestines  presenting  a  hemorrhagic 
inflammation. 

Guinea-pigs  and  sheep  are  immune.  Mice  and  rabbits  are 
affected  in  the  same  manner  as  the  fowls. 

Immunity. — Pasteur,  by  injecting  different-aged  cultures  into 
fowls,  produced  in  them  only  a  local  inflammation,  and  they 
were  then  immune.  But  as  the  strength  of  these  cultures  could 
not  be  estimated,  many  fowls  died  and  the  healthy  ones  were 
endangered  from  the  intestinal  excretions,  which  is  the  chief 
manner  of  infection  naturally,  the  feces  becoming  mixed  with 
the  food. 

Bacteria  of  Hemorrhagic  Septicemia  (Hueppe) . — Under 
this  heading  Hueppe  has  gathered  a  number  of  bacteria  very 
similar  to  the  bacillus  of  chicken  cholera,  differing  from  it  and 
each  other  but  very  little.  They  have  been  described  by  various 
observers  and  found  in  different  diseases. 

The  bacteria  of  this  group  color  themselves  strongly  at  the 
poles,  giving  rise  to  the  dumb-bell  shape.  They  do  not  take 
the  Gram  stain;  they  are  without  spores,  and  do  not  liquefy 
gelatin. 

They  have  been  placed  in  three  general  divisions: 
'Wild  plague  (Hueppe). 
German  swine  plague  (Loffler,  Schiitz). 


First  division. 


Rabbit  septicemia. 


Ox  plague  (Oresti-Armanni) . 
^  Steer  plague  (Kitt) . 
The  bacteria  of  the  first  division  are  not  motile,  do  not  grow 
on  potato,  and  are  found  scattered  through  the  blood-vessels. 
A  local  reaction  is  uncommon. 

C  American  swine  plague  (Billings) . 

c        j  7-  •  •       I  French  swine  plague  (Cornil  and  Chante- 
becond  division.  <  . 

messe) . 

L  Frog  plague  (Eberth) . 
Here  the  bacteria  are  motile.    They  grow  on  potatoes  and 


BACTERIA   PATHOGENIC    FOR   ANIMALS,    NOT   FOR   MAN   2OI 

are  similar  to  the  typhoid  bacillus  in  gelatin.  They  form 
small  embolic  processes  in  the  capillaries.  They  cause  only 
a  local  disturbance  in  rabbits  when  subcutaneously  injected. 
An  acid  fermentation  is  produced  in  milk. 

™,  •   ,  7-  •  •         f  Hog-cholera  (Salmon). 
Third  division.  {  c    6..  ,  .        '   T    .      , 

\  Swedish  swine  plague  (Lelander). 

The  bacteria  of  this  third  division  are  very  motile.  The 
hog-cholera  bacilli  lie  in  the  spleen  and  other  organs  in  small 
masses  like  the  typhoid  bacillus. 


Fig.  122. — Bacillus  of  swine- plague  (from  photograph  by  F.  A.  de  Schweinitz). 

Rabbits  die  in  four  to  eight  days  without  any  local  disturb- 
ance. The  growth  on  potato  is  strong. 

The  Swedish  swine-plague  bacillus  occupies  a  position 
between  that  of  hog-cholera  and  Bacillus  coli  communis. 

The  various  swine-plague  bacilli  are  but  little  active  in  fowls, 
differing  thus  widely  from  the  chicken-cholera  bacillus. 

Bacillus  of  Erysipelas  of  Swine  (Lb  filer,  Schtitz) ; 
Schweinerotlaufbacillus  (German) ;  Rouget  du  Pore 
(French) . — Origin. — Found  in  the  spleen  of  an  erysipelatous 
swine  by  Loffler  in  1885. 


202          ESSENTIALS  OF  BACTERIOLOGY 

Form. — One  of  the  smallest  forms  of  bacilli  known;  very  thin, 
seldom  longer  than  i  //,  looking  at  first  like  little  needle-like 
crystals.  Spores  have  not  been  found. 

Properties. — They  are  motile;  do  not  liquefy  gelatin. 

Growth  at  ordinary  temperature,  very  slowly,  and  the  less 
oxygen,  the  better  the  growth. 

Gelatin  Plate. — On  third  day  little  silver-gray  specks,  seen 
best  with  a  dark  background,  coalescing  after  a  while,  pro- 
ducing a  clouding  of  the  entire  plate. 

Stab-cultures. — In  a  few  days  a  very  light,  silvery-like  cloud- 
ing, which  gradually  involves  the  entire  gelatin;  held  up 
against  a  dark  object,  it  comes  plainly  into  view. 

Staining. — All  ordinary  dyes  and  Gram's  method  also. 

Tissue  sections  stained  by  Gram's  method  show  the  bacilli  in 
the  cells,  capillaries,  and  arterioles  in  great  numbers. 

Pathogenesis. — Swine,  mice,  rabbits,  and  pigeons  are  sus- 
ceptible; guinea-pigs  and  chickens,  immune. 

When  swine  are  infected  through  food  or  by  injection,  a  tor- 
pidity develops  with  diarrhea  and  fever,  and  on  the  belly  and 
breast  red  spots  occur  which  coalesce,  but  do  not  give  rise  to 
any  pain  or  swelling.  The  animal  dies  from  exhaustion  in 
twenty-four  to  forty-eight  hours.  In  mice  the  lids  are  glued 
together  with  pus. 

At  the  autopsy  the  liver,  spleen,  and  glands  are  enlarged  and 
congested,  little  hemorrhages  occurring  in  the  intestinal  mucous 
membrane  and  that  of  the  stomach. 

Bacilli  are  found  in  the  blood  and  in  all  the  viscera. 

One  attack,  if  withstood,  protects  against  succeeding  ones. 

Immunity. — Has  also  been  attained  by  injecting  vaccines  of 
two  separate  strengths. 

Bacillus  Murisepticus  (Koch) ;  Mouse  Septicemia. — 
Origin. — Found  in  the  body  of  a  mouse  which  had  died  from 
injection  of  putrid  blood,  and  described  by  Koch  in  1878. 

Form. — Differs  in  no  particular  from  the  bacillus  of  swine 
erysipelas,  excepting  that  it  is  a  very  little  shorter,  making  it 
the  smallest  known  bacillus.  Spores  have  been  found,  the  cul- 
tures exactly  similar  to  those  of  swine  erysipelas. 


BACTERIA   PATHOGENIC   FOR  ANIMALS,    NOT   FOR   MAN   203 

The  pathologic  actions  are  also  similar.  Field-mice  are 
immune,  whereas  for  house  and  white  mice  the  bacillus  is  fatal 
in  two  to  three  days. 

Micrococcus  of  Mai  de  Pis  (Nocard). — Gangrenous 
mastitis  of  sheep. 

Origin. — In  the  milk  and  serum  of  a  sheep  sick  with  the 
"mat  de  pis." 


Fig.  123. — Bacillus  of  mouse  septicemia,  from  the  blood  of  a  mouse  (x  1000) 
(Frankel  and  Pfeiffer). 

Form. — Very  small  cocci  seldom  in  chains. 

Properties. — Immotile;  liquefying  gelatin. 

Growth. — Growth  occurs  best  between  20°  and  37°  C.,  is  very 
rapid,  and  irrespective  of  oxygen. 

Plates  of  Gelatin. — White  round  colonies,  some  on  the  surface 
and  some  in  the  deeper  strata,  with  \owpower,  appearing  brown 
surrounded  by  a  transparent  areola. 


204  ESSENTIALS   OF   BACTERIOLOGY 

Stab-culture. — Very  profuse  along  the  needle-track,  in  the 
form  of  a  cone  after  two  days,  the  colonies  having  gathered  at 
the  apex. 

Potato. — A  dirty  gray,  not  very  abundant,  layer,  somewhat 
viscid. 

Staining. — With  ordinary  methods;  also  Gram's  method. 

Pathogenesis. — If  a  pure  culture  is  injected  into  the  mammary 
gland  of  sheep,  a  "mal  de  pis'7  is  produced  which  causes  the 
death  of  the  animal  in  twenty-four  to  forty-eight  hours.  The 
breast  is  found  edema tous,  likewise  the  thighs  and  perineum; 
the  mammae  very  much  enlarged,  and  at  the  nipples  a  blue- 
violet  coloration.  The  spleen  is  small  and  black;  other  animals 
are  less  susceptible.  In  rabbits  abscesses  at  the  point  of  infec- 
tion, but  no  general  affection. 

Bacillus  Alvei  (Cheshire  and  Cheyne) ;  Bacillus  melit- 
tophtharus  (Cohn) .— Origin.— In  foul-brood  of  bees. 

Form. — Slender  rods,  with  round  and  conical  pointed  ends; 
very  large  oval  spores,  the  rod  becoming  spindle-shaped  when 
they  appear. 

Properties. — Motile,  liquefying  gelatin  rapidly. 

Growth. — Grows  best  between  20°  C.  and  37°  C.,  very 
slowly;  aerobic. 

Gelatin  Plates. — Small  grooves  are  slowly  formed,  which  unite 
so  as  to  form  a  circle  or  pear-shaped  growth,  from  which  linear 
grooves  again  start. 

Stab-culture. — Grows  first  on  surface,  then  gradually  along 
the  needle-track,  long  processes  shooting  out  from  the  same, 
clouding  the  gelatin.  Later,  air-bubbles  form  like  the  cholera 
culture,  and  in  two  weeks  the  whole  gelatin  liquefies. 

Staining. — Do  not  take  anilin  dyes  very  well.  Gram's 
method  is,  however,  applicable. 

Pathogenesis. — If  a  pure  culture  is  spread  over  the  honey- 
comb containing  bee  larvae,  or  if  bees  are  fed  upon  infected 
material,  foul-brood  disease  will  occur.  Mice,  if  injected,  die 
in  a  few  hours.  Edema  around  the  point  of  infection,  and 
many  bacilli  contained  in  the  edematous  fluid,  otherwise  no 
changes. 


BACTERIA  PATHOGENIC   FOR  ANIMALS,   NOT  FOR  MAN   205 

Bacterium  Termo  (Cohn). — This  was  a  name  given  to  a 
form  of  microorganism  found  in  decomposing  albuminous 
material,  and  was  supposed  to  be  one  specific  germ.  Hauser, 
in  1885,  found  three  different  distinct  bacilli  which  he  grouped 
under  the  common  name  of  proteus,  which  have  the  putrefy- 
ing properties  ascribed  to  Bacillus  termo. 

Proteus  Vulgaris. — Origin. — In  putrid  animal  matter,  in 
the  feces,  and  in  water. 

Form. — Small  rods,  slightly  curved,  of  varying  lengths,  often 
in  twisted  chains,  having  long  cilia  or  flagella. 

Properties. — Very  motile,  and  very  soon  liquefying  gelatin; 
forms  hydrogen  sulphid  gas;  causes  putrefaction  in  meat. 

Growth. — Growth  very  rapid,  best  at  24°  C.;  is  facultative 
aerobic. 

Gelatin  Plates. — Yellowish-brown,  irregular  colonies,  with 
prolongations  in  every  direction,  forming  all  sorts  of  figures;  an 
impression  preparation  shows  these  spider-leg  processes  to  con- 
sist of  bacilli  in  regular  order. 

Stab-culture. — The  gelatin  soon  liquid,  a  gray  layer  on  the 
surface,  but  the  chief  part  of  the  culture  in  small  crumbs  at  the 
bottom. 

Pathogenesis. — Rabbits  and  guinea-pigs  injected  subcutane- 
ously  die  quickly;  a  form  of  toxemia,  hemorrhagic  condition  of 
lungs  and  intestines  present.  When  neurin  is  injected  previ- 
ously, the  animals  do  not  die.  This  ptomain  is  supposed  to  be 
generated  by  the  Proteus  vulgaris. 

Proteus  Mirabilis  (Hauser). — Differs  from  Proteus  vul- 
garis in  that  the  gelatin  is  less  rapidly  liquefied.  Found  also 
in  putrid  material. 

Proteus  Zenkeri  (Hauser). — Does  not  liquefy  gelatin; 
otherwise  similar  to  the  other  two. 


206  ESSENTIALS   OF   BACTERIOLOGY 

CHAPTER  XXII 
YEASTS  AND  MOLDS 

IN  works  on  bacteria  these  true  fungi,  yeasts  and  molds,  are 
usually  considered.  They  are  so  closely  related  to  bacteria,  and 
so  often  contaminate  the  culture-media,  and  are  so  similar  in 
many  respects,  that  a  description  is  almost  a  necessity. 

But  there  are  several  thousand  varieties,  and  we  cannot 
attempt  to  describe  even  all  the  more  important  ones.  It 
will  answer  our  purpose  to  detail  a  few  of  the  more  common 
kinds,  and  give  the  principal  features  of  the  different  orders. 


Fig.  124. — Yeast-cells  (x  500)  (Frankel  and  Pfeiffer). 

Saccharomycetes  or  yeasts  increase  through  budding;  the 
spores  are  attached  to  the  mother-cell  like  a  tuber  on  a  potato. 

Yeasts  are  the  cause  of  alcoholic  fermentation  in  the  saccha- 
roses. A  description  of  the  most  common  ones  will  suffice. 

Saccharomyces  Cerevisiae  (Torula  Cerevisiae). — This  is 
the  ordinary  beer-yeast. 


YEASTS    AND   MOLDS  207 

Form. — Round  and  oval  cells;  a  thin  membrane  inclosing  a 
granular  mass,  in  which  usually  can  be  seen  three  or  four  irreg- 
ular-shaped spores.  When  these  become  full  grown,  they  pass 
through  the  cell- wall  and  form  a  daughter-cell.  Sometimes  long 
chains  are  produced  by  the  attached  daughter-cells. 

Growth. — They  can  be  cultivated  as  bacteria  are  in  bouillon, 
but  grow  best  in  beer. 

There  are  several  varieties  of  beer-yeast,  each  one  giving  a 
characteristic  taste  to  the  beer.  Brewers,  by  paying  special 
attention  to  the  nutrient  media,  cultivate  yeasts  which  give  to 
their  beers  individual  flavors. 

Mixed  yeast  gives  rise  to  a  poor  quality  of  beer. 

Saccharomyces  Rosaceus;  S.  Niger;  S.  Albicans.— These 
yeasts  are  found  in  the  air;  and  instead  of  producing  alcoholic 
fermentation,  they  give  rise  to  a  pigment  in  the  culture-media. 
They  grow  upon  gelatin,  which  they  do  not  liquefy. 

Saccharomyces  Mycoderma. — This  yeast  forms  a  mold-like 
growth,  or  skin,  on  the  surface  of  fermented  liquids,  but  does  not 
cause  any  fermentation  itself.  It  forms  the  common  "mold" 
on  wine,  preserves,  and  "sour-krout." 

Pathogenic  Yeasts. — A  number  of  workers  have  interested 
themselves  in  experiments  with  yeasts  in  their  relation  to  dis- 
ease; and  under  the  name  of  blastomycetes ,  Sanfelice  has  grouped 
yeasts  that  produce  tumors  resembling  epitheliomata ;  and  he 
has  tried  to  prove  that  the  so-called  animal  parasites  found  in 
malignant  growths,  and  variously  known  as  coccidia  and  sporo- 
zoa,  are  yeasts.  These  are,  however,  protozoa. 

Oi'dium. — A  form  which  seems  to  be  the  bridge  between  the 
yeast  and  the  molds  is  the  oidium.  Sometimes  it  resembles 
the  yeasts,  sometimes  the  molds,  and  often  both  forms  are 
found  in  the  same  culture.  Several  are  pathogenic  for 
man. 

Oi'dium  Lactis. — Origin. — In  sour  milk  and  butter. 

Form. — The  branches  or  hyphae  break  up  into  short,  rod- 
like  spores.  No  sporangium,  as  in  molds. 

Growth. — In  milk  it  appears  as  a  white  mold. 

Artificially  cultured  on  gelatin  plates,  or  milk-gelatin  plates, 


208  ESSENTIALS  OF  BACTERIOLOGY 

it  forms  satin-like,  star-shaped  colonies,  which  slowly  liquefy. 
Under  the  microscope  the  form  of  the  fungus  is  well  seen. 

Agar  Stroke  Culture. — The  little  stars,  very  nicely  seen  at  first; 
then  the  culture  becomes  covered  with  them,  causing  a  smeared 
layer  to  appear  over  the  whole  surface,  with  a  sour  odor. 

Properties. — The  milk  is  not  changed  in  any  special  way.  It 
is  not  pathogenic  for  man  or  animals.  It  is  found  when  the 
milk  begins  to  sour. 

Oi'dium  Albicans  (Soor;  Thrush  Fungus,  Langenbeck, 
1839). — Origin. — Mucous  membrane  of  the  mouth,  especially 
of  infants. 

Form. — Taken  from  the  surface  of  the  culture,  a  form  like 
yeasts;  but  in  the  deeper  layers,  mycelia  with  hyphae  occur. 

Growth. — Not  liquefying;  snow-white  colonies  on  gelatin 
plates. 

Stab-culture. — Radiating  yellow  or  white  processes  spring 
from  the  line  made  by  the  needle,  those  near  the  surface  having 
oval  ends. 

Potatoes. — The  yeast  form  develops  as  thick  white  colonies. 

Bread-mash. — Snow-white  veil  over  the  surface. 

Pathogenesis. — In  man  the  parasitic  thrush,  or  "  white 
mouth,"  is  caused  by  this  fungus.  In  the  white  patches  the 
spores  and  filaments  of  this  microbe  can  be  found.  Rabbits 
receiving  an  intravenous  injection  perish  in  twenty-four  to 
forty-eight  hours,  the  viscera  being  filled  with  mycelia. 

Blastomycosis  or  Oi'dium  Mycosis. — A  skin  disease 
described,  in  1894,  by  Gilchrist,  and  since  then  by  other 
writers,  is  due  to  a  fungus  which  resembles  yeast,  and  which 
has  been  called  a  blastomyces;  but  Ophuls  and  Ricketts  term 
it  an  o'idium,  and  the  former  calls  the  parasite  Oi'dium  cocci- 
dioides. 

Form. — The  fungus  increases  by  budding,  but  in  culture- 
media  it  may  resemble  a  mold  or  o'idium. 

Pathogenesis. — Small  abscesses  form  in  wart-like  lesions, 
which  extend  over  large  areas  of  the  skin,  becoming  later 
on  systemic  and  invading  lungs  and  kidneys;  abscesses  and 
nodules  form  in  these  organs. 


YEASTS   AND    MOLDS  2OQ 

True  Molds. — Fliigge  has  made  five  distinct  divisions  of 
molds.  It  will,  however,  serve  our  purpose  to  classify  those 
to  be  described  under  three  headings :  Penicillium,  Mucor,  and 
As  per  gill  us. 

Penicillium  Glaucum. — Origin. — The  most  widely  dis- 
tributed of  all  molds,  found  wherever  molds  can  exist. 

Form. — From  the  mycelium,  hyphae  spring  which  divide  into 
basidia  (branches) ,  from  which  tiny  filaments  arise  (sterigmata) , 
arranged  like  a  brush  or  tuft.  On  each  sterigma  a  little  bead 


Fig.  125. — Penicillium  glaucum  (x  500)  (Frankel  and  Pfeiffer). 

or  conidium  forms,  which  is  the  spore.  In  this  particular  fun- 
gus the  spores  in  mass  appear  green. 

Growth. — It  develops  only  at  ordinary  temperatures,  forming 
thick,  grayish-green  molds  on  bread-mash.  At  first  these 
appear  white,  but  as  soon  as  the  spores  form,  the  green  pre- 
dominates. Gelatin  is  liquefied  by  it. 

Mucor  Mucedo. — Next  to  the  Penicillium  glaucum,  this  is 
the  most  common  mold.  Found  in  horse-dung,  in  nuts  and 
apples,  in  bread  and  potatoes,  as  a  white  mold. 

Form. — The  mycelium  sends  out  several  branches,  on  one  of 
14 


2IO 


ESSENTIALS  OF  BACTERIOLOGY 


which  a  pointed  stem  is  formed  which  enlarges  to  form  a  globu- 
lar head,  a  spore-bulb,  or  sporangium.  The  spore-bulb  is  par- 
titioned off  into  cells  in  which  large  oval  spores  lie.  When  the 
spores  are  ripe,  a  cap  forms  around  the  bulb,  the  walls  break 
down,  and  the  wind  scatters  the  spores,  leaving  the  cap  or 
"columella"  behind. 

Growth. — Takes  place  at  higher  temperatures  on  acid  media. 
It  is  not  pathogenic. 

Achorion  Schb'nleinii.  Trichophyton  Tonsurans.  Mi- 
crosporon  Furfur. — These  three  forms  are  similar  to  each 
other  in  nearly  every  particular,  and  resemble  in  some 


Fig.  126. — Achorion  Schonleinii  (after  Kaposi). 

respects  the  Oidium  lactis,  in  other  ways,  the  mucors.  The 
first  one,  Achorion  Schonleinii,  was  discovered  by  Schonlein 
in  1839,  m  favus,  and  is  now  known  as  the  direct  cause  of  this 
skin  disease. 

Origin. — Found  in  the  scaly  crusts  of  favus. 

Form. — Similar  to  Oidium  lactis. 

Growth. — Is  very  sparse.  On  gelatin  round  white  masses 
inclosed  by  a  zone  of  liquefied  gelatin. 

In  milk  it  is  destroyed. 

Pathogenesis. — Causes  favus  in  man. 

Trichophyton  Tonsurans.— Found,  in  1854,  by  Bazin,  in 
tinea. 


YEASTS    AND    MOLDS  211 

Form. — Similar  to  the  achorion  or  favus  fungus. 

Growth. — Somewhat  more  rapid  than  the  favus,  and  the  gela- 
tin quickly  liquefied.  Old  cultures  are  of  an  orange-yellow 
color.  Colonies  have  a  star-shaped  form. 

Patho genesis. — Herpes  tonsurans  and  the  various  tinese  are 
produced  by  this  fungus. 

Microsporon  Furfur. — Found  in  tinea  versicolor,  almost 
identical  with  the  above,  forms  dry  yellow  spots,  usually  on  the 
chest,  in  persons  suffering  from  wasting  diseases. 


Fig.  127. — Aspergillus  fumigatus  (x  500)  (Frankel  and  Pfeiffer). 

Aspergillus  Glaucus. — Origin. — In  saccharine  fruits. 

Form. — The  hypha  has  formed  upon  its  further  end  a  bulb, 
from  which  pear-shaped  sterigmata  arise  and  bear  upon  their 
ends  the  conidia  or  spores. 

Growth. — Best  upon  fruit-juices.  Non-pathogenic.  The 
mold  is  green.  Aspergillus  flavus  has  the  tufts  and  spores  of  a 
yellow  color. 

Aspergillus  Fumigatus. — Is  pathogenic  for  rabbits  when 
injected  into  them.  At  the  autopsy  their  viscera  are  found 
filled  with  the  mold. 


212 


ESSENTIALS   OF    BACTERIOLOGY 


Examination  of  Yeasts  and  Molds.— Yeasts  and  molds  are 
best  examined  in  the  unstained  condition.  A  small  portion  of 
the  colony  rubbed  up  with  a  mixture  of  alcohol  and  a  few  drops 
of  liquor  ammonia;  of  this,  a  little  is  brought  upon  the  glass 
slide,  covered  with  a  drop  of  glycerin;  and  the  cover- glass 
pressed  upon  it.  If  the  preparation  is  to  be  saved,  the'  cover- 
glass  is  secured  by  ringing  around  the  edges  with  varnish  or 
cement.  Yeasts  take  methylene-Uue  stain  very  well. 

Cladothrices  and  Streptothrices.— The  streptothrix  and 
cladothrix  groups  are  classed  with  the  higher  bacteria,  but 


Fig.  128. — Cladothrix  dichomata  from  well-water  (one-twelfth  oil-immersion. 
Fuchsin  stain)  (author's  specimen). 

their  exact  status  is  still  undetermined.  They  may  be  consid- 
ered as  representing  the  transition  from  the  bacteria  to  the 
lower  fungi. 

Streptothrix  or  Cladothrix  Actinomyces  (Ray-fungus)  .— 
Actinomycosis  is  a  disease  caused  in  man  and  cattle  by  this 
organism,  which  is  commonly  found  in  grain,  particularly 
barley.  It  is  probable  that  several  varieties  of  the  parasite  can 
produce  the  characteristic  lesions.  It  has  been  discovered  in 
all  countries  and  in  various  organs  of  the  body,  although  its 
place  of  election  is  about  the  lower  jaw,  where  it  tends  to  form 
hard,  ulcerating  abscesses,  affecting  other  organs  secondarily. 

Form. — In  the  granular  masses  of  an  abscess  cylindric  fila- 


YEASTS   AND    MOLDS  213 

ments  are  matted  tagether,  and  radiating  outward  from  this 
zone  are  club-shaped  branches,  as  the  petals  of  an  aster.  In  the 
center  of  the  granule  are  numerous  cocci-like  bodies,  and  some 
of  the  ovoid  or  club-shaped  hyphae  lie  detached  from  the 
clusters.  Through  cultivation  it  was  found  that  the  ovules  give 
rise  to  filaments,  and  they  then  form  the  ovules  again. 

Cultivation. — At  38°  C.  on  glycerin-agar  in  a  period  of  one  to 
two  weeks,  pointed  scales  about  the  size  of  a  millet-seed,  center 


Fig.  129. — Actinomyces  granule  crushed  beneath  a  cover-glass,  showing 
radial  striations  in  the  hyaline  masses.  Preparation  not  stained;  low  magnify- 
ing power  (Wright  and  Brown). 

dry  and  prominent,  margins  hyaline,  composed  only  of  fila- 
ments, short  and  long,  massed  together,  but  no  clubbed  forms. 

By  some  the  clubs  are  considered  the  spore  organs;  by  others, 
they  are  thought  to  be  encapsulated  or  thickened  filaments. 

Patho genesis. — When  a  portion  of  the  growth  obtained  in 
eggs  was  injected  into  the  abdominal  cavity  of  a  rabbit,  actino- 
mycotic  processes  developed  upon  the  peritoneum. 

It  usually  gains  access  to  the  living  body  through  a  wound  in 
the  gum  or  some  caries  of  the  teeth.  A  new  growth  is  formed, 
ulceration  being  first  set  up. 


214 


ESSENTIALS  OF  BACTERIOLOGY 


The  new  tissue,  composed  of  round-cells,  then  undergoes 
softening,  purulent  collections  form,  and  the  normal  structure 
is  destroyed. 

The  usual  seat  is  in  the  maxillary  bones,  but  the  fungus  has 
been  found  in  the  lungs,  tonsils,  intestines,  and  various  other 

,_    organs  in  man  and  cattle. 

Examination. — Well     seen 
in    the   unstained  condition. 
jjjjj    From  the  pus  or  scraping  a 
small  portion   is   taken  and 
squeezed  upon  the  glass  slide; 
IJH    if  calcareous  matter  is  pres- 
js||    ent,  a  drop  of  nitric  acid  will 
dissolve  the  same. 

Glycerin  will  preserve  the 
preparation. 

Staining.  —  Cover  -glass 
specimens  stained  best  with 
Gram's  method.  Tissue  sec- 
tions should  be  stained  as 
follows : 

Ziehl's  carbol-fuchsin,  ten 
minutes.  Rinse  in  water. 

Concentrated  alcoholic 
solution  of  picric  acid,  five 
minutes.  Rinse  in  water. 

Alcohol,  50  per  cent.,  fif- 
teen minutes.     Alcohol  abso- 
lute, clove-oil,  balsam. 
The  rays  stained  red,  the  tissue  yellow. 
Streptothrix  Madurae  (Vincent). — Origin. — Found  in  the 
disease  known  as  Madura  foot,  or  mycetoma,  an  ulceration 
affecting  the  feet,  especially  of  individuals  living  in  the  tropics. 
Two  varieties,  the  pale  and  the  black,  have  been  described. 

Form. — Branched    filaments    resembling    the    actinomyces 
Streptothrix  in  the  mycelia.     Spores  are  seen. 
Cultivation. — In  liquid  media  containing  vegetable  infusions 


Fig.  130. — Streptothrix  Madurae  in  a 
section  of  diseased  tissue  (Vincent). 


YEASTS   AND   MOLDS  215 

growth  occurs  best.  Temperature  of  37°  C.  most  suited. 
The  colonies  near  the  surface  become  colored  red. 

Agar. — Glazed  colonies,  at  first  colorless,  then  rose-colored, 
about  the  size  of  a  pea,  with  the  central  part  umbilicated  and 
pale.  Gradually  the  rose  color  fades. 

Acid  Potato. — A  slow  and  meager  growth. 

Pathogenesis. — Only  local  reaction  has  been  caused  by  inocu- 
lation in  animals.  In  man  the  disease  usually  follows  a  slight 
injury  and  attacks  the  leg  or  foot,  slowly  forming  a  nodular 
growth,  which  in  the  course  of  months  or  a  year  begins  to 
soften  and  ulcerate,  and  with  the  seropus  are  discharged 
numerous  little  granules,  some  black,  some  pink,  containing 
mycelia.  The  limb  becomes  much  deformed,  the  tissue 
vascularized,  and  the  degenerated  area  filled  with  the  strepto- 
thrix  filaments. 

Staining. — The  organism  itself  stained  with  ordinary  stains. 
Gram's  method  for  the  tissue. 

Streptothrix  Farcinica  (Nocard) ;  Bovine  Farcin  du 
Boeuf . — Origin. — A  disease  affecting  cattle  and  giving  rise  to 
tubercle-like  lesions  in  the  lungs,  liver,  and  spleen.  Common 
in  France. 

Form. — Small  interwoven  mass  of  threads  arranged  in  tufts 
found  in  the  centers  of  the  tubercles. 

Culture. — At  body-temperature  in  various  media. 

Bouillon. — Colorless  masses,  irregular  in  size  and  shape. 

Agar  and  Gelatin. — Small,  rounded,  opaque  colonies,  thicker 
at  the  periphery. 

Potato. — Rapid  growth  of  pale-yellow,  dry  scales,  consisting 
of  many  spores. 

Pathogenesis. — Pure  cultures  introduced  into  the  peritoneum 
of  guinea-pigs  give  rise  in  nine  to  twenty  days  to  tubercle-like 
lesions.  Subcutaneous  injections  cause  abscesses  with  sec- 
ondary involvement  of  the  lymphatics,  ending  in  recovery. 
Dogs,  horses,  and  rabbits  are  immune. 

Staining. — Wright's  double  stain  for  tissues;  also  Gram's. 


2l6  ESSENTIALS   OF   BACTERIOLOGY 

CHAPTER  XXIII 
EXAMINATION  OF  AIR,  SOIL,  AND  WATER 

Air. — Many  germs  are  constantly  found  in  the  atmosphere 
about  us.  Bacteria  unaided  do  not  rise  into  the  air  and  fly 
about;  they  usually  become  mixed  with  small  particles  of  dirt 
or  dust  and  are  moved  with  the  wind.  The  more  dust,  the 
more  bacteria,  and,  therefore,  the  air  in  summer  contains  a 
greater  number  than  the  air  in  winter,  and  all  the  other  differ- 
ences can  be  attributed  to  the  greater  or  less  quantity  of  dust 
and  wind. 

Methods  of  Examination. — The  simplest  method  is  to 
expose  a  glass  or  dish  covered  with  gelatin  in  a  dust-laden 
atmosphere  or  in  the  place  to  be  examined.  In  the  course  of 
twenty-four  to  forty-eight  hours  colonies  will  be  seen  formed 
wherever  a  germ  has  fallen.  But  this  method  will  not  give  any 
accurate  results  in  regard  to  the  number  of  bacteria  in  a  given 
space;  for  such  a  purpose  somewhat  more  complicated  methods 
are  needed,  so  that  a  certain  amount  of  air  can  come  in  contact 
with  the  culture-media  at  a  certain  regulated  rate  of  speed. 

Hesse's  Method. — This  is  the  oldest  and  most  useful  of 
the  various  methods  in  vogue. 

A  glass  cylinder,  70  cm.  long  and  3.5  cm.  in  diameter,  is 
covered  at  one  end  by  two  rubber  caps,  the  inner  one  having 
a  hole  in  its  center  10  mm.  in  diameter;  and  at  the  end  B,  a 
rubber  cork  fits  in  the  cylinder;  through  this  cork  a  glass  tube 
10  mm.  in  diameter  passes,  which  is  plugged  at  both  ends  with 
cotton.  The  cylinder  and  fittings  are  first  washed  in  alcohol 
and  sublimate  and  then  placed  for  one  hour  in  the  steam- 
chamber. 

Removing  the  cork  of  the  cylinder,  50  c.c.  of  sterile  gelatin 
in  a  fluid  condition  are  introduced  and  rolled  out  on  the  sides 
of  the  tube,  after  the  manner  of  Esmarch,  leaving  a  somewhat 
thicker  coating  along  the  under  side  of  the  cylinder.  The 
aeroscope,  as  the  cylinder  and  its  fittings  are  called,  is  placed 


EXAMINATION    OF   AIR,    SOIL,    AND   WATER  217 

upon  an  ordinary  photographer's  tripod  and  the  glass  tube, 
which  passes  through  the  rubber  cork,  connected  with  an 
aspirator,  the  cotton  having  first  been  removed  from  its  outer 
end.  The  aspirator  consists  of  two  ordinary  wash-bottles 
connected  with  each  other  by  a  rubber  tube,  C.  They  are 
attached  to  the  tripod  with  a  small  hook,  one  above  the  other, 
the  upper  one  half  filled  with  water  and  slightly  tilted. 


Fig.  131 . — Hesse's  apparatus  for  collecting  bacteria  from  the  air  (McFarland). 

When  the  apparatus  is  wanted,  the  outer  rubber  cap  at  the 
end  A  of  the  aeroscope  is  removed;  the  air  can  then  pass 
through  the  small  hole  in  the  other  cap,  and  the  germs  fall 
upon  the  gelatin  in  the  tube,  the  cotton  in  the  small  glass  tube 
at  the  other  end  preventing  the  germs  from  getting  out.  The 
aspirator  is  set  in  use  by  tilting  the  upper  bottle  so  that  the 
water  flows  into  the  lower;  this  creates  suction  and  draws  the 
air  through  the  aeroscope. 


218 


ESSENTIALS   OF   BACTERIOLOGY 


The  amount  entering  is  estimated  by  the  capacity  of  the 
wash-bottle,  the  rate  at  which  it  enters  depending  upon  the 
rate  of  the  flow  of  water,  which  can  be  regulated. 


Fig.  132. — Petri's  sand-filter  for  air- 
examination  (McFarland). 


Fig.  133. — Sedgwick's  expanded  tube 
for  air-examination  (McFarland). 


Hesse  advises  for  rooms  and  closed  spaces  i  to  5  liters,  at  the 
rate  of  two  minutes  a  liter,  and  for  open  spaces,  10  to  20  liters, 
at  four  minutes  a  liter.  Plate  cultures  can  be  made  from  the 
colonies,  which  develop  in  eight  to  ten  days  in  the  cylinder. 

Petri's  Method.— The  air  is  pumped  or  sucked  through 
sand-filters,  and  the  sand  then  mixed  with  gelatin. 


EXAMINATION   OF   AIR,    SOIL,    AND    WATER  2IQ 

Sand  is  sterilized  by  heating  to  redness,  and  while  still 
warm  placed  in  test-tubes,  which  are  then  plugged.  (Sand 
which  has  been  passed  through  a  sieve  with  meshes  0.25  mm. 
wide  is  the  kind  required.)  A  glass  tube  9  cm.  long  is  provided 
with  two  portions  of  sand,  each  3  cm.  long  and  J  cm.  apart, 
little  plates  of  brass  gauze  keeping  the  portions  in  position. 

The  tube  and  its  contents  are  now  sterilized  in  a  hot-air  oven 
at  150°  C.,  the  ends  having  first  been  plugged  with  cotton. 

One  end  of  the  tube  is  then  fitted  with  a  rubber  cork  through 
which  passes  a  glass  tube,  which  is  connected  with  an  aspirator 
(a  hand-pump  with  a  known  capacity) . 

If  a  100  liters  of  air  pass  through  the  tube  in  fifteen  min- 
utes, the  germs  should  all  be  arrested  in  the  first  sand-filter. 
And  when  the  filters  are  removed  and  thoroughly  mixed  with 
gelatin,  each  filter  for  itself,  there  should  be  no  colonies  devel- 
oped from  the  second  filter,  i.  e.,  the  one  nearest  the  aspirator. 

Sedgwick-Tucker  Method. — A  special  form  of  tube  is  used, 
called  an  aerobioscope.  It  consists  of  a  neck  2.5  cm.  in  length, 
an  expanded  portion  15  cm.  long,  and  a  long  narrow  tube  of  15 
cm.  After  sterilization  the  tube  is  partly  filled  with  granulated 
sugar,  which  is  the  filtering  material.  By  means  of  a  vacuum 
gage  and  an  air-pump,  or  ordinary  aspirating  bottles,  the  vol- 
ume of  air  passing  through  the  apparatus  can  be  determined. 
After  the  air  has  been  passed  through,  the  sugar  is  gently 
shaken  from  the  narrow  tube  into  the  expanded  portion,  and 
20  c.c.  of  liquefied  gelatin  is  poured  in.  The  sugar  dissolves, 
and  the  mixture  is  then  rolled  on  the  inner  side  of  the  glass 
as  an  Esmarch  tube.  This  part  of  the  apparatus  is  divided  into 
squares  to  make  the  counting  of  colonies  easy.  The  aerobio- 
scope is  very  highly  recommended. 

Varieties  Found  in  Air. — The  only  pathogenic  bacteria 
found  with  any  constancy  are  the  Staphylococcus  aureus  and 
citreus;  but  any  bacterium  can,  through  accident,  be  lifted  into 
the  atmosphere,  and  in  certain  places  may  be  always  found — 
the  Bacillus  tuberculosis,  for  example,  in  rooms  where  many 
consumptives  are  living. 

Typhoid  fever,  influenza,  pneumonia,  and  diphtheria  may  be 


220  ESSENTIALS   OF   BACTERIOLOGY 

conveyed  through  the  air  by  those  who  are  infected  by  coughing 
and  expectorating. 

Non-pathogenic. — The  micrococci  predominate.  Sarcinae, 
yeasts,  and  molds  constantly  contaminate  cultures. 

In  the  ordinary  habitations  the  average  number  of  germs  to 
the  liter  of  air  does  not  exceed  five. 

Around  water-closets,  where  one  would  imagine  a  great  num- 
ber to  exist,  owing  to  the  undisturbed  condition  of  the  air,  but 
few  will  be  found. 

Examination  of  Water. — The  bacteriologic  examination  of 
water  is  to-day  of  as  much  importance  as  the  chemical  analysis, 
and  must  go  hand  in  hand  with  it. 

A  water  containing  thousands  of  germs  to  the  cubic  centi- 
meter is  far  less  dangerous  than  one  containing  but  two  germs, 
if  one  of  these  two  be  a  typhoid  bacillus.  It  is  not  the 
number  that  proves  dangerous,  it  is  the  kind. 

If  a  natural  water  contains  more  than  500  germs  to  the  cubic 
centimeter,  it  were  well  to  examine  its  source. 

Bacteriology  performs  the  greatest  service  in  testing  the 
devices  which  are  intended  to  render  water  fit  for  drinking. 

As  a  diagnostic  aid,  the  examination  is  of  but  little  use.  An 
epidemic  of  typhoid  fever  occurs,  the  water  is  suspected,  an 
examination  is  undertaken;  but  the  days  of  incubation  and  the 
days  passed  before  the  water  is  analyzed  have  given  the  typhoid 
germs,  if  any  had  been  present,  ample  time  to  disappear,  since 
in  water  that  contains  other  bacteria  they  live  but  a  few  days 
only.  Again,  the  water  tested  one  day  may  be  entirely  free  and 
the  next  day  contain  a  great  number,  and  before  the  typhoid 
germ  can  be  proved  to  be  present  in  that  particular  water  the 
epidemic  may  be  past.  Human  sewage  contamination  is 
determined  by  finding  the  colon  bacillus,  and  if  this  is  found 
in  the  course  of  an  epidemic  of  typhoid  the  water  containing 
it  may  well  be  suspected  as  being  the  cause. 

Purity  of  Waters. — The  purest  water  we  have  is  the  natural 
spring-water — water  that  has  slowly  filtered  its  way  through 
various  layers  of  gravel  and  sand  and  comes  finally  clear  and 
sparkling  from  the  ground.  It  is  without  germs;  but  let  such 


EXAMINATION    OF   AIR,    SOIL,    AND   WATER  221 

a  water  stand  walled  up  in  cisterns  or  wells,  it  becomes  as 
surface  water,  open  to  all  sorts  of  impurities,  and  the  bacterial 
nature  of  it  changes  every  moment. 

Artesian  or  Driven  Well. — The  driven  well  will  secure  to  a 
certain  extent  a  pure  water.  It  is  the  only  form  of  well  or 
cistern  that  will  insure  this,  since  the  water  does  not  become 
stagnant  in  it;  but  it  may  connect  with  an  outhouse,  the  soil 
being  very  loose,  allowing  the  products  of  germs  of  refuse 
water  to  find  their  way  into  the  well.  The  casing  may  not  be 
water-tight  and  surface  water  can  be  sucked  in. 

Filtered  Water. — Dangerous  as  surface  water  is,  the  greater 
quantity  used  is  such,  the  inhabitants  of  larger  towns  and 
cities  using  chiefly  the  rivers  and  other  large  waters  which 
course  near  them  for  drinking  purposes.  A  purification  or 
filtration  can,  in  a  certain  measure,  render  these  waters  harm- 
less. 

Filtration  is  carried  on  on  a  large  scale  in  the  water-works 
of  cities  and  towns,  and  bacteriologic  examination  is  here  of 
great  service  to  determine  if  a  water,  which  has  been  filtered 
and  may  have  a  very  clear  appearance,  and  give  no  harmful 
chemical  reaction,  is  entirely  free,  or  nearly  so,  from  germs; 
in  other  words,  if  the  filter  is  a  germ-filter  or  not;  daily  tests 
are  necessary  in  order  to  insure  safety. 

Charcoal  sponge  and  asbestos,  the  materials  formerly  in 
use,  are  objectionable  because  germs  readily  develop  on  them 
and  clog  them,  so  that  they  require  frequent  renewal.  In  very 
large  filters,  sand  and  gravel  give  the  best  results;  the  number 
of  germs  in  a  cubic  centimeter  is  reduced  to  forty  or  fifty  and 
kept  at  that  number.  This  is  a  very  pure  water  for  a  city  water, 
though,  as  we  stated  before,  not  a  safe  one,  for  among  those 
forty  germs  very  dangerous  ones  may  be  found.  It  is  then 
necessary  for  the  users  to  refilter  the  water,  before  drinking  it, 
through  a  material  which  will  not  allow  any  germs  to  pass,  or, 
in  the  presence  of  an  epidemic,  to  boil  all  water  used  for 
drinking  purposes. 

Pasteur-Chamberland  Filter. — This  very  perfect  filter 
consists  of  a  piece  of  polished  porcelain  in  the  form  of  a 


222  ESSENTIALS   OF   BACTERIOLOGY 

cylinder  closed  at  one  end  and  pointed  at  the  other.  It  is 
placed  in  another  cylinder  of  glass  or  rubber,  and  the  pointed 
portion  connected  with  a  bottle  containing  the  water,  or  directly 
with  the  faucet  of  the  water-pipe.  The  water  courses  through 
the  porcelain  very  slowly  and  comes  out  entirely  free  from 
germs;  pipe-clay,  bisque,  infusorial  earth,  and  kaolin  are  also 
perfect  niters.  The  only  disadvantage  is  the  long  time  it  takes 
for  the  water  to  pass  through.  Pressure  is  used  to  accelerate 
the  passage  in  the  form  of  an  aspirator  or  air-pump. 


Fig.  134. — Flask  fitted  with  porcelain  bougie  for  filtering  large  quantities  of 

fluid. 

The  force  of  the  hydrant  water  is  also  sufficient  to  produce  a 
steady,  small  stream. 

These  porcelain  cylinders  can  easily  be  sterilized  and  the 
pores  washed  out. 

All  the  cylinders  or  bougies  are  not  germ  proof,  so  that  they 
must  be  tested,  and  most  of  them  must  be  cleaned  every  fourth 
day,  or  they  will  allow  germs  to  pass  through. 

Boiling  as  a  Means  o)  Purifying. — When  such  a  filter  cannot 
be  obtained,  the  only  alternative  is  to  boil  all  the  water  to  be 
used  for  drinking;  and  this  should  especially  be  done  in  times 
of  typhoid  and  cholera  epidemics. 


EXAMINATION  OF   AIR,    SOIL,    AND   WATER  223 

Varieties  Found. — The  usual  kinds  found  are  non-patho- 
genic, but,  as  is  well  known,  typhoid,  cholera,  and  dysen- 
tery are  principally  spread  through  drinking-water,  and  many 
other  germs  may  find  their  way  into  the  water.  Some  of  the 
common  varieties  give  rise  to  fluorescence  or  produce  pigment. 

Eisenberg  gives  100  different  varieties  as  ordinarily  found. 
Other  intestinal  diseases  also  are  supposed  to  be  water  borne, 
and  the  presence  of  the  Bacillus  coli  communis  means  sewage 
contamination.  Ice  supplies  require  the  same  supervision  as 
water  supplies,  for  many  bacteria,  like  the  typhoid  bacillus, 
retain  their  vitality  for  weeks  after  freezing. 

Methods  of  Examination.— Since  the  germs  rapidly  mul- 
tiply in  stagnant  water,  an  examination  must  not  be  delayed 
longer  than  possible  after  the  water  has  been  collected.  Every 
precaution  must  be  taken  in  the  way  of  cleanliness  to  prevent 
contamination;  sterilized  flasks  with  glass  stoppers,  pipets,  and 
plugs  must  be  at  hand,  and  the  gelatin  tubes  be  inoculated  on 
the  spot.  If  this  cannot  be  done,  the  sample  should  be  packed 
in  ice  until  it  arrives  at  the  laboratory.  If  it  is  necessary  to 
send  the  sample  by  rail,  the  bottle  containing  the  sample  should 
be  wrapped  in  sterilized  cloth  or  the  neck  covered  with  tin- 
foil and  the  bottles  placed  in  tin  boxes  (about  4  ounces — 100 
c.c. — is  sufficient  for  bacterial  analysis),  and  then  packed  in 
cotton  or  paper  to  prevent  breakage  and  surrounded  by  plenty 
of  ice  until  it  reaches  its  destination.  As  soon  as  it  arrives  at 
the  laboratory  the  sample  is  placed  in  a  sterilized  glass  flask, 
and  the  flask  then  closed  with  a  sterile  cotton  plug.  A  sterilized 
pipet  is  then  dipped  into  the  flask,  and  i  c.c.  of  the  water  with- 
drawn and  added  to  a  tube  of  gelatin,  the  gelatin  being  in  a 
fluid  condition.  To  a  second  tube,  J  c.c.  is  added.  The  tubes 
are  then  shaken  so  as  to  thoroughly  mix  the  water  with  the 
gelatin,  and  then  poured  upon  wide  glass  plates  or  porous 
covered  Petri  dishes,  one  plate  for  each  tube;  the  plates  are 
then  placed  in  the  moist  chamber  and  in  two  or  three  days 
examined.  A  temperature  of  18°  to  20°  C.  is  best.  Many 
water-bacteria  are  hindered  by  higher  degrees  of  heat.  If  the 
germs  are  equally  divided,  there  should  be  one-half  the  number 


224  ESSENTIALS    OF    BACTERIOLOGY 

on  one  plate  that  there  is  on  the  other;  thus  the  i  c.c.  serves  as 
control. 

Water  that  is  very  rich  in  germs  requires  dilution  with  ster- 
ilized water  fifty  to  one  hundred  times.  Fewer  colonies  will 
be  found  on  agar  than  on  gelatin,  even  at  the  same  temperature. 

Special  Media  and  Preparation. — Sodium  chlorid  must  not 
be  used  in  the  preparation  of  media  for  water  analysis.  The 
reaction  of  most  culture-media  should  be  +i  per  cent,  to 
phenolphthalein. 

Sugar  broths  should  be  neutral,  and  must  be  sterilized  care- 
fully in  steam  and  not  overheated,  to  prevent  inversion  of  the 
sugar. 

Examination  for  Bacillus  Coli  and  Sewage  Bacteria. — 
Instead  of  examining  for  typhoid  bacilli,  sewage  contamination 
is  best  indicated  by  the  presence  of  the  colon  group  of 
organisms. 

The  committee  of  the  Public  Health  Association  recom- 
mends the  following  procedure : 

Two  Methods. — Method  a. — Preparation  of  an  agar  plate 
with  a  known  volume  of  water,  using  lactose  litmus-agar  and 
incubating  at  40°  C.  Bacillus  coli  will  show  its  presence  by  red 
colonies  (acid  fermentation  of  the  sugar) ;  further  testing  is  then 
needed  to  fully  identify. 

Method  b. — Cultivation,  at  40°  C.,  of  a  measured  quantity 
of  water  in  a  fermentation  tube  containing  a  sugar  broth. 
If  gas  appears,  a  portion  of  the  liquid  is  plated  as  in  method  a. 

Additional  Details. — If  in  twenty-four  hours  no  red  colonies 
appear  in  the  agar-lactose  litmus  Petri  dishes,  Bacillus  coli  is 
considered  absent,  providing  the  sample  was  a  polluted  one, 
so  that  the  bacilli,,  if  present,  would  be  in  a  concentrated  form. 
Only  i  or  2  c.c.  of  water  can  be  used,  because  the  ordinary 
water-bacteria  spread  rapidly  and  contaminate  the  other  bac- 
teria. 

If  acid-forming  colonies  are  found,  five  or  six  are  fished  for 
subcultures  on  slanted  agar,  in  fermentation  tubes,  milk, 
gelatin,  peptone  solution,  and  nitrate  broth. 

If  the  water  is  not  strongly  contaminated,  an  underground 


EXAMINATION   OF  AIR,    SOIL,    AND   WATER          225 

water,  for  instance,  or  a  mountain  stream,  the  better  way  is  to 
inoculate  two  or  three  lactose  or  dextrose  bouillon  fermentation 
tubes  and  place  in  an  incubator  at  40°  C.  Note  the  presence 
of  gas,  if  any,  at  the  end  of  twelve,  twenty-four,  thirty-six, 
and  forty-eight  hours.  If  no  gas  forms,  sewage  bacteria  are 
absent. 

If  gas  forms,  plate  at  once  a  portion  of  the  sediment  as 
above  on  lactose  litmus-agar.  Test  the  other  fermentation 
tubes  for  acidity,  and  the  nature  of  the  gas,  whether  any,  and 
how  much  is  absorbed  by  a  2  per  cent,  solution  of  sodium 
hydroxid.  Bacillus  coli  should  produce  between  30  and 
70  per  cent,  of  gas,  of  which  about  one-third  is  CO2  and 
is  absorbed  by  the  alkali;  the  remainder  is  hydrogen.  The 
other  broth  culture  can  be  tested  for  the  presence  or  absence  of 
unfermented  sugar,  for  the  color  reaction  of  Rivas. 

Quantitative  Tests. — The  number  of  acid  colonies  in  i  c.c. 
and  in  5  c.c.  of  water  is  taken  as  a  measure  of  pollution,  together 
with  the  total  number  of  colonies  of  all  bacteria  present.  Thus 
in  i  c.c.  on  the  gelatin  plate  at  20°  C.  there  may  be  fifty  colonies; 
on  the  agar  plate  at  37°  C.  ten  colonies,  five  of  which  were 
acid-formers,  or  presumably  Bacillus  coli. 

To  count  the  colonies  which  develop  upon  the  plates,  a 
special  apparatus  has  been  designed,  known  as — 

Wolfhiigel's  Apparatus.— A  glass  plate  divided  into 
squares,  each  a  centimeter  large,  and  some  of  these  subdivided. 
This  plate  is  placed  above  the  gelatin  plate  with  the  colonies, 
and  the  number  in  several  quadrants  taken,  a  lens  being  used 
to  see  the  smaller  ones. 

The  Petri  saucers  can  be  used  instead  of  plates,  and  an 
apparatus  on  the  Wolfhugel  plan  can  be  obtained  to  count 
the  colonies.  It  is  best  to  count  all  the  colonies  on  the  plate 
or  dish. 

Diagnostic  Points  of  Colon  Bacillus.— Microscopic.— 
Non-spore-bearing  motile  bacillus. 

Gelatin. — Non-liquefactive. 

Dextrose  Broth. — Fifty  per  cent,  gas;  one-third  absorbed, 
CO2;  two-thirds,  hydrogen. 
15 


226          ESSENTIALS  OF  BACTERIOLOGY 

Milk  (litmus)  coagulated  in  forty-eight  hours  and  rendered 
acid;  litmus  colored  red. 

Peptone  Solution. — Production  oj  Indol. — (A  peptone  solution 
tube  is  inoculated  with  the  culture  and  kept  together  with  a 
control  four  days  at  37°  C.  Then  2  drops  of  concentrated 
sulphuric  acid  and  i  centimeter  of  a  o.oi  per  cent,  solution 
of  sodium  nitrite  are  added.  The  appearance  of  a  pink  color 
at  the  end  of  thirty  minutes  denotes  the  presence  of  indol.) 

Presumptive  Test. — If  a  water  from  a  well  or  spring  produces 
gas  in  the  sugar  broth  and  forms  acid  colonies  on  litmus- 
lactose  agar,  the  presumption  is  strong  that  there  is  sewage 
contamination.  If  gas-production  continues  in  a  series  of 
samples  carefully  collected  for  several  days  or  weeks,  there  can 
be  no  doubt  of  a  contamination,  and  especially  if  the  well  or 
spring  is  protected  from  surface  water.  Algae  which  grow  in 
service  pipes,  reservoirs,  and  deep  wells  may  give  rise  to 
non-acid  gas  fermentation,  but  all  well-water  that,  without 
further  testing,  forms  acid  colonies  on  litmus-agar  lactose 
plates  and  ferments  sugar  broth,  is  open  to  suspicion,  and  if 
there  is  evidence  of  the  presence  of  typhoid  fever  or  diarrheal 
diseases,  the  water  should  be  boiled  and  subjected  to  care- 
ful analysis  daily.  There  may  be  serious  contamination 
and  the  chemical  tests  show  no  appreciable  increase  in  the 
chlorids. 

Bacterial  Treatment  of  Sewage.— Where  sewage  is  to  be 
rendered  innocuous  before  being  allowed  to  flow  into  streams, 
the  process  of  nature  has  been  imitated  by  the  construction  of 
septic  tanks  in  which  the  sewage  remains  excluded  from  the 
air  and  subject  to  the  action  of  the  anaerobic  bacteria  present 
in  the  sewage.  The  organic  nitrogen  is  reduced,  and  com- 
pounds of  hydrogen  and  sulphur  are  formed.  The  effluent 
is  then  filtered  through  coke-beds,  where  the  aerobic  bacteria 
assist  in  further  purification. 

Sewage  is  also  treated  by  sedimentation  with  alum  and 
filtration  of  the  effluent  over  larger  beds,  or  allowed  to  percolate 
through  the  soil,  which  is  thereby  enriched  and  utilized  for 
agriculture. 


EXAMINATION   OF    AIR,    SOIL,    AND   WATER  227 

The  Examination  of  the  Soil. — The  upper  layers  of  the 
soil  contain  a  great  many  bacteria,  but  because  of  the  difficulty 
in  analyzing  the  same,  the  results  are  neither  accurate  nor  con- 
stant. The  principal  trouble  lies  in  the  mixing  of  the  earth 
with  the  nutrient  medium;  little  particles  of  ground  will  cling 
to  the  walls  of  the  tube,  or  be  embedded  in  the  gelatin,  and 
may  contain  within  them  myriads  of  bacteria.  As  with  water, 
the  soil  must  be  examined  immediately  or  very  soon  after  it  is 
collected,  the  bacteria  rapidly  multiplying  in  it. 

When  the  deeper  layers  are  to  be  examined,  some  precautions 
must  be  taken  to  avoid  contamination  with  the  other  portions  of 
the  soil.  One  method,  very  laborious  and  not  often  practical, 
is  to  dig  a  hole  near  the  spot  to  be  examined  and  take  the  earth 
from  the  sides  of  this  excavation. 

Frankel's  Borer. — Frankel  has  devised  a  small  apparatus 
in  the  form  of  a  borer,  which  contains  near  its  lower  end  a  small 
cavity,  which  can  be  closed  up  by  turning  the  handle,  or  opened 
by  turning  in  the  opposite  direction. 

It  is  introduced  with  the  cavity  closed,  and  when  it  is  at  the 
desired  depth,  the  handle  is  turned,  the  earth  enters  the  cavity, 
the  handle  again  turned,  incloses  it  completely,  and  the  borer  is 
then  withdrawn. 

The  earth  can  then  be  mixed  with  the  gelatin  in  a  tube,  and 
this  gelatin  then  rolled  on  the  walls  of  the  tube  after  the  man- 
ner of  Esmarch,  or  it  can  be  poured  upon  a  glass  plate,  and 
the  colonies  developed  so. 

Another  method  is  to  wash  the  earth  with  sterilized  water, 
and  the  water  then  mixed  with  the  gelatin,  as  many  of  the 
germs  are  taken  up  by  the  water. 

The  roll-cultures  of  Esmarch  give  the  best  results,  many  of 
the  varieties  usually  found  being  anaerobic. 

Animals  inoculated  with  the  soil  around  Berlin  die  almost 
always  of  malignant  edema,  and  with  that  of  some  other  towns 
invariably  of  tetanus.  Many  of  the  germs  found  are  nitrogen 
formers  and  play  a  great  role  in  the  economy  of  the  soil. 

Nitrifying  organisms  are  found  in  the  superficial  layers  of  the 
earth.  Organic  matters  found  in  sewage  and  in  the  fecal 


228  ESSENTIALS    OF   BACTERIOLOGY 

evacuations  of  animals  form  the  basis  for  their  activity,  whereby 
nitrates,  ammonias,  and  nitric  acid  result.  The  nitrogen 
necessary  for  the  growing  plant  is  thus  produced.  The 
nitromonas  of  Winogradsky  belongs  to  this  group.  The  soil 
tends  to  destroy  ordinary  disease-bacteria  in  a  short  time,  but 
spores  may  remain  dormant  for  a  number  of  years  as  the 
spores  of  anthrax. 

The  Bacteria  of  Milk  and  Other  Foods.— Milk  as  secreted 
is  sterile,  but  at  every  step  in  its  passage  from  the  cow  to  the 
consumer  it  is  liable  to  contamination.  Even  the  lower  portion 
of  the  teat  is  a  source  of  infection,  owing  to  the  presence  of 
stagnated  milk  from  the  former  milking,  and,  as  milk  ready  for 
consumption  usually  contains  thousands  to  millions  of  bacteria 
to  the  cubic  centimeter,  sterilization  or  pasteurization  and  super- 
vision of  the  dairies  should  always  be  carried  out  on  milk  used 
for  infant  feeding. 

A  standard  milk  should  be  free  from  pus  and  should  not 
contain  more  than  10,000  bacteria  to  the  cubic  centimeter. 

Leukocytes  are  normally  found  in  milk,  and  only  when 
their  number  exceeds  one  million  and  pyogenic  organisms  are 
also  present  can  pus  be  said  to  exist.  Pasteurization  of  unclean 
milk  is  sometimes  more  dangerous  as  a  food  than  untreated 
milk,  because,  by  preventing  the  action  of  lactic-acid  formers, 
other  bacteria  are  permitted  to  develop  and  produce  pathogenic 
toxins. 

Pure  Milk. — A  pure  milk  is  one  that  is  obtained  from  a 
healthy  cow,  well  groomed,  in  a  clean  room,  by  a  healthy, 
clean  person,  in  clean  cans  or  bottles,  and  transported  to  the 
consumer  in  as  short  time  as  possible  without  further  hand- 
ling, keeping  the  container  in  the  mean  time  at  a  low  tem- 
perature and  protected  from  the  air.  Such  treatment  is  safer 
than  any  form  of  sterilization. 

Foods  as  a  Source  oj  Injection. — Foods  eaten  after  little  or 
no  cooking,  such  as  fruits,  salads,  and  the  like,  and  also 
oysters,  are  possible  sources  of  bacterial  diseases,  and  the  not 
infrequent  so-called  ptomain  poisoning  observed  after  the 


ORGANS   AND   CAVITIES   OF  THE  HUMAN  BODY      22C) 

consumption  of  ice-cream,  sausage,  canned  meats,  etc.,  is  the 
result  of  the  action  of  bacteria  or  their  products. 

Oysters  and  fish  from  sewage-polluted  waters  have  produced 
typhoid.  Vegetables  grown  in  manured  ground  or  sprinkled 
with  polluted  water  may  be  a  possible  source  of  disease. 
The  practise  of  exposing  meats  and  other  food  to  street  dust 
and  flies  is  no  doubt  responsible  for  some  disease. 


CHAPTER  XXIV 

BACTERIOLOGIC  EXAMINATION  OF  THE   ORGANS  AND 
CAVITIES  OF  THE  HUMAN   BODY 

THE  body,  on  account  of  its  constant  contact  with  the  sur- 
rounding air,  is  necessarily  exposed  to  infection,  and  we  would 
be  likely  to  find  on  the  skin  and  in  the  oral,  anal,  and  nasal 
cavities  the  varieties  of  microorganisms  commonly  around  us. 
Through  the  water  and  food  the  body  is  also  contaminated; 
but  some  organisms  by  predilection  inhabit  the  mouth,  intes- 
tine, and  other  cavities,  and  form  there  a  flora  distinctly  their 
own. 

The  Skin. — The  majority  of  microorganisms  met  with  on  the 
skin  are  non-pathogenic,  although  underneath  the  nails  and  in 
the  hair,  pus-forming  microorganisms  often  occur,  producing 
sometimes  serious  abscesses  on  other  parts  of  the  body. 

In  the  sweat-glands  and  the  sebaceous  glands  various  organ- 
isms have  been  found.  The  Staphylococcus  epidermidis  albus 
of  Welch  is  present  normally. 

In  foul-smelling  perspiration  of  the  feet  Rosenbach  found 
Saprogenes  No.  II,  which  is  pathogenic  for  rabbits. 

Micrococcus  cereus  albus  and  flavus,  Diplococcus  liquefa- 
ciens  albus  and  flavus,  Staphylococcus  pyogenes  aureus,  and 
Streptococcus  pyogenes  are  found  underneath  the  nails. 

In  eczema,  Diplococcus  albicans  tardus,  Diplococcus  citreus 


230  ESSENTIALS    OF   BACTERIOLOGY 

liquefaciens,  Diplococcus  flavus  liquefaciens,  and  Ascobacillus 
citreus. 

In  colored  sweat,  Micrococcus  haematoides,  Bacillus  pyocya- 
neus. 

A  diplococcus  is  found  in  acute  pemphigus. 

The  lepra  bacillus,  the  tubercle  bacillus  in  lupus,  and  the 
typhoid  bacillus  in  the  eruption  of  typhoid  fever  are  a  few  of 
the  specific  germs  found  on  the  skin. 

The  Conjunctiva.— The  micrococcus  of  trachoma,  the 
Koch- Weeks  bacillus,  considered  to  be  the  specific  cause  of 
acute  catarrhal  conjunctivitis,  or  "pink  eye,"  and  the  Bacillus 
xerosis,  are  special  germs  found  on  the  conjunctiva;  the  other 
varieties  of  air-  and  water-organisms,  and  those  usually 
present  on  the  skin,  are  also  found.  Loffler's  bacillus  and  the 
pneumococcus  have  been  found  in  some  forms  of  conjunctivitis. 
The  Koch-Weeks  bacillus  is  the  most  contagious. 

A  special  diplobacillus,  known  as  the  bacillus  of  Morax- 
Axenfeld,  produces  a  stubborn  form  of  conjunctivitis. 

The  Mouth. — The  mouth  is  a  favorite  seat  for  the  develop- 
ment of  bacteria.  The  alkaline  saliva,  the  particles  of  food 
left  in  the  teeth,  the  decayed  teeth  themselves,  all  furnish 
suitable  soil  for  their  growth. 

Quite  a  number  of  germs  have  been  isolated  and  their  prop- 
erties partly  studied.  Many  have  some  connection  with  the 
production  of  caries  of  the  teeth,  as  Miller  has  well  shown  in 
his  careful  studies.  The  Leptothrix  buccalis,  found  in  nearly 
all  mouths,  is  a  long  chain  or  filamentous  bacillus  which  stains 
blue  with  iodin.  It  was  formerly  considered  the  cause  of  tartar 
on  the  teeth. 

The  Spirillum  sputigenum,  Spirochaeta  dentium,  Micrococcus 
gingivae  pyogenes,  Bacillus  dentalis  viridans,  Bacillus  pulpae 
pyogenes,  micrococcus  of  sputum  septicemia,  and  Micrococcus 
salivarus  septicus  are  a  few  of  the  germs  cultivated  by  Miller 
and  Biondi  from  the  mouth.  Besides  these,  the  pneumo- 
bacteria,  diphtheria  bacillus,  and  tubercle  bacillus  are  often 
met  with,  the  first  two  in  the  mouths  of  healthy  persons.  The 
expired  air  in  quiet  respiration  is  free  from  bacteria,  but  in 


ORGANS   AND   CAVITIES   OF    THE   HUMAN   BODY      231 

coughing,  sneezing,  etc.,  large  numbers  of  organisms  are  vio- 
lently ejected  and  the  atmosphere  about  tubercular  patients 
is  always  saturated  with  tubercle  bacilli. 

Ear. — In  the  middle  ear  of  newborn  infants  no  pathogenic 
organisms  were  found,  but  quite  a  number  of  non-pathogenic 
ones.  In  affections  of  the  ear  the  pneumobacillus  and  the 
Staphylococcus  pyogenes  are  most  frequent. 

When  the  streptococcus  is  present  in  acute  suppurations, 
there  is  great  danger  of  mastoiditis.  In  chronic  otitis  the  gas- 
forming  bacteria,  as  well  as  Bacillus  pyocyaneus,  is  often 
found. 

Nasal  Cavity. — The  nasal  secretion,  containing  as  it  does 
dead  cells  and  being  alkaline  in  reaction,  forms  a  good  soil  for 
the  growth  of  germs. 

Diplococcus  coryzae,  Micrococcus  nasalis,  Bacillus  fcetidus 
ozaenae,  Bacillus  striatus  albus  et  flavus,  Bacillus  capsulatus 
mucosus,  and  Vibrio  nasalis  are  some  of  the  organisms  de- 
scribed by  various  observers. 

Stomach  and  Intestine. — The  secretion  of  the  stomach  is 
in  its  normal  state  not  a  favorable  soil  for  the  development  of 
bacteria,  yet  some  germs  resist  the  action  of  the  gastric  juice 
and  flourish  in  it.  When  the  acids  of  the  stomach  are  dimin- 
ished in  quantity  or  absent  altogether,  the  conditions  for  the 
growth  of  bacteria  are  more  favorable.  The  alimentary  canal 
of  the  newborn  infant  is  sterile,  but  in  a  few  hours  micro- 
organisms begin  to  appear. 

Some  gastric  bacteria  normally  present  are  Sarcina  ventric- 
uli,  Bacterium  lactis  aerogenes,  Bacillus  subtilis,  Bacillus 
amylobacter,  Bacillus  megaterium. 

The  intestinal  organisms  are  more  numerous,  and  the 
mucous  lining  of  the  intestines  and  the  secretions  there  present 
are  favorable  to  germ-growth. 

Bacillus  geniculatus,  Boas  considers  a  sign  of  carcinoma  of 
the  stomach,  and  is  always  present,  he  claims,  when  the  con- 
tents contain  lactic  acid. 

Some  investigators  consider  digestion  dependent  on  microbic 
activity,  but  experiments  with  animals  have  shown  that  life 


232  ESSENTIALS    OF   BACTERIOLOGY 

and  digestion  can  proceed  in  a  perfectly  sterile  condition. 
Food  and  air  sterilized  will  not  develop  bacteria  in  the  feces. 

In  the  feces  of  the  young  a  great  many  bacteria  have  been 
found  that  are  supposed  to  stand  in  close  relation  with  the 
intestinal  disorders  common  to  nurslings.  The  majority  of 
bacteria  usually  present  in  the  intestines  are  non-pathogenic. 
The  following  varieties  may  be  met  with  in  the  feces:  Micro- 
coccus  aerogenes,  Bacillus  subtilis,  Bacillus  butyricus,  Bacillus 
putrificus  coli,  Bacillus  lactis  aerogenes,  Bacillus  coli  commune, 
Bacillus  subtiliformis,  and  the  bacteria  of  cholera,  dysentery, 
and  typhoid,  besides  many  yeast-cells. 

Genito -urinary  Passages. — In  vaginal  secretion  Bumm 
has  been  able  to  find  a  number  of  organisms,  some  of  which 
closely  resemble  the  gonococcus;  thus,  there  is  the  Diplococcus 
subflavus,  Micrococcus  lacteus  faviformis,  Diplococcus  albicans 
amplus,  and  the  vaginal  bacillus. 

In  the  urethra  of  healthy  persons  bacteria  are  sometimes 
found,  usually  having  entered  from  the  air. 

In  the  normal  secretions  around  the  prepuce  a  bacillus  called 
the  smegma  bacillus  has  been  discovered,  and  it  is  considered 
identical  with  the  so-called  syphilis  bacillus  of  Lustgarten. 

In  urethral  pus  a  number  of  diplococci  other  than  the  gono- 
cocci  have  been  isolated. 

From  the  urine  itself  a  great  number  of  bacteria  have  been 
obtained,  but  mostly  derived  from  the  air,  finding  in  the  urine 
a  suitable  soil. 

Microorganisms  of  the  Blood. — Many  of  the  bacteria 
described  in  the  body  of  this  book  are  found  in  the  blood  of  the 
animal  they  infect;  thus,  anthrax  bacilli  are  always  found 
in  the  blood,  but  tubercle  bacilli  seldom,  if  ever,  enter  this 
secretion. 

When  animals  are  subcutaneously  injected  with  pneumo- 
cocci  they  are  found  in  large  quantities  in  the  blood.  The 
diseases  of  a  hemorrhagic  nature  affecting  fowls  and  swine 
usually  show  the  presence  of  bacteria  in  the  vascular 
system. 

Bacteria  may  be  recovered  from  the  blood  in  all  forms  of 


ANTISEPTICS  AND  ANTISEPSIS  233 

septic  infection,  such  as  general,  sepsis,  malignant  endocarditis, 
puerperal  sepsis,  and  typhoid  fever. 

Method  of  Examination. — A  drop  of  blood  is  spread  on  a 
cover-glass  and  stained  with  the  ordinary  dyes;  but  in  order 
to  eliminate  the  coloring-matter  of  the  red  corpuscles  and  bring 
the  stained  bacteria  more  prominently  into  view,  Gunther 
recommends  that  the  blood,  after  drying  and  fixing,  should  be 
rinsed  in  a  dilute  solution  of  acetic  acid  (i  to  5  per  cent.). 
The  hemoglobin  is  thereby  extracted,  and  the  corpuscles  appear 
then  only  as  faint  outlines. 

Instead  of  "fixing"  by  heat,  Canon  employs  alcohol  for  five 
minutes,  especially  in  staining  for  influenza  bacilli,  which  have 
been  detected  in  the  blood. 

This  method,  however,  requires  the  presence  of  enormous 
numbers  of  bacteria  in  order  to  succeed,  and  the  plan  com- 
monly employed  consists  in  making  "  blood  cultures."  As 
large  a  quantity  of  blood  as  possible — never  less  than  10  c.c. — 
is  taken  from  a  superficial  vein,  the  median  basilic,  for  example, 
by  means  of  a  sterile  antitoxin  syringe,  a  small  incision  being 
made  through  the  skin  over  the  vein  in  order  to  avoid  skin 
infection.  The  blood  so  obtained  is  immediately  transferred  to 
culture-tubes,  which  are  then  studied  in  the  customary  manner. 


CHAPTER  XXV 
ANTISEPTICS  AND  ANTISEPSIS 

A  germicide  is  an  agent  capable  of  destroying  bacterial  life. 

An  antiseptic  solution  or  substance  is  one  that  can  inhibit 
or  prevent  the  growth  of  bacteria  without  necessarily  destroying 
them. 

A  disinfectant  must  be  germicidal. 

A  deodorant  may  have  no  germicidal  or  antiseptic  properties. 


234          ESSENTIALS  OF  BACTERIOLOGY 

Preservatives  are  substances  which  prevent  fermentation, 
but  they  are  not  always  germicides. 

In  considering  the  value  of  a  germicide,  the  strength  in 
which  it  acts  is  the  main  consideration.  Some  very  weak 
chemicals  will  inhibit  and  destroy  the  growth  of  bacteria  if 
used  in  sufficiently  concentrated  solutions.  Some  bacteria  will 
die  in  an  acid  media;  others  are  destroyed  by  too  much  alkali. 
Some  bacteria  are  very  readily  destroyed  in  pure  cultures,  but 
are  resistant  to  a  considerable  degree  in  the  body  tissues. 
Again,  a  germicide  may  be  ideal  in  laboratory  experiments, 
but  wholly  impractical  at  the  clinic. 

Germicides  are  tested  by  action  in  various  dilutions  or  in 
gaseous  form  on  threads  impregnated  with  virulent  and  spore- 
forming  organisms.  The  length  of  time  is  noted  that  it  takes 
to  destroy  anthrax  bacilli  or  pyogenic  organisms. 

The  infected  material  is  subjected  to  the  solution  and  then 
inoculated  on  media  and  compared  with  control,  or  tested  for 
virulence  on  animals.  Spore-forming  organisms  are  very 
resistant  to  the  most  potent  agents. 

Heat  is  perhaps  the  best  general  germicide.  For  all  articles 
that  can  be  subjected  to  boiling  or  the  direct  flame  there  is 
no  safer  agent. 

Superheated  steam,  or  steam  under  pressure,  is  now  in 
general  use  in  sterilizing  surgical  dressings  and  instruments, 
and  requires  less  time  than  ordinary  steam. 

The  salts  of  metals  of  high  atomic  weights  come  next  in  order. 
Bichlorid  of  mercury  and  cyanid  of  mercury  are  the  most 
powerful  of  chemical  germicides,  but  in  the  human  body  they 
can  be  used  in  dilute  solutions  only,  and  in  contact  with  highly 
albuminous  solutions,  insoluble  and  inert  albuminates  are 
liable  to  form,  lessening  the  germicidal  value.  A  i  :  200 
solution  combined  with  an  acid  will  destroy  the  spores  of 
anthrax  in  one  hour,  but  much  weaker  solutions  will  destroy 
the  anthrax  bacilli  in  the  blood,  and  for  all  practical  purposes 
a  i  :  2000  solution  is  sufficient,  destroying  bacterial  life  in  a 
few  minutes. 


ANTISEPTICS  AND  ANTISEPSIS  235 

Phenol  in  5  per  cent,  solution  will  destroy  most  of  the  bac- 
teria in  less  than  five  minutes. 

Formaldehyd^  in  gaseous  form  or  in  a  liquid  spray,  is  a  very 
efficient  germicide,  and  from  the  fact  that  it  is  not  destructive 
to  fabrics  or  paper  has  come  into  general  use  as  a  disinfectant. 
In  combination  with  potassium  permanganate  or  in  suitable 
generators  it  is  employed  in  houses  after  infectious  diseases. 
It  has  no  effect  on  insects,  and  where  it  is  necessary  to  destroy 
these,  other  agents,  known  as  insecticides,  must  be  used  in  con- 
nection with  the  gas.  The  gas  should  be  in  a  moist  state — from 
6  to  1 6  ounces  for  an  ordinary  room  are  needed;  the  room 
should  be  made  as  air-tight  as  possible,  and  the  gas  evolved  as 
speedily  as  possible. 

In  the  permanganate  method  8  ounces  (by  weight)  of 
potassium  permanganate  crystals  are  placed  in  a  large  tin 
vessel  ten  times  the  capacity  of  the  disinfectant  used.  One 
pint  of  formaldehyd  solution  is  quickly  poured  over  the 
crystals.  Formaldehyd  gas  is  thereby  generated  at  once. 
This  will  produce  enough  gas  for  disinfection  of  1000  cubic 
feet. 

Sulphur  dioxid,  or  sulphurous  acid  gas,  is  a  germicide  and 
insecticide,  and  is  much  used  in  disinfecting  after  yellow  fever 
and  malaria.  It  is  obtained  by  burning  sulphur  in  a  pan  over 
water,  and  about  i  pound  to  a  room  is  necessary. 

Alcohol,  iodin,  chlorin,  potassium  permanganate,  hydrogen 
dioxid,  the  salts  of  silver,  lead,  and  zinc,  salicylic  acid,  boric  acid, 
anilin  dyes  (methyl-violet  and  methylene-blue) ,  naphthalin,  and 
creosols  are  a  few  of  the  substances  in  use  as  antiseptics 
and  germicides  in  surgery.  Their  power  varies  with  the 
strength  of  the  solution  and  all  have  limitations. 

In  surgical  operations  more  dependence  is  placed  to-day 
on  securing  and  maintaining  a  germ-free  or  a  septic  condition 
than  on  the  attempt  to  destroy  germ  life  by  chemicals.  The 
irritation  of  antiseptics  in  some  instances  prevents  the  natural 
defense  forces  (phagocytes)  from  acting,  and  in  abdominal 
operations,  where  no  pus  has  been  encountered,  the  blood- 
serum  or  normal  salt  solution  is  alone  used. 


236  ESSENTIALS    OF    BACTERIOLOGY 

Sterilization  of  Hands,  etc. — It  has  been  shown  by  elaborate 
experiments  that  the  skin,  the  hair,  and  clothing  harbor  many 
bacteria,  some  of  a  pathogenic  nature.  The  surgeon  who  is 
anxious  to  secure  good  results  should  carefully  attend  to  his 
toilet;  the  use  of  operating  gowns,  rubber  gloves,  operating 
shoes,  face  guards  is  now  universal.  The  toilet  of  the  hands 
of  the  surgeon  is  as  important  as  that  of  the  field  of  operation, 
but  with  the  use  of  rubber  gloves  the  painstaking  directions  as 
to  the  employment  of  a  half-dozen  or  more  cleansing  agents 
and  germicides  are  no  longer  followed. 

Soap  is  an  efficient  germicide,  the  lye  being  in  most  cases 
powerful  enough  to  prevent  the  growth  of  germs. 

Filtration. — In  the  laboratory  and  on  a  larger  scale  in  the 
management  of  water-works,  filtration  is  a  method  of  steriliza- 
tion, acting  as  it  does  by  mechanically  separating  bacteria 
from  a  solution. 


CHIEF    CHARACTERISTICS 

CHIEF  CHARACTERISTICS 
PAKT  L— 


Name. 

Genus. 

Biology. 

Product. 

ACETI. 

ACIDI  LACTICI. 

Bacillus. 
Bacillus. 

Short  motile  rods   in 
zooglcea  ;  aerobic. 
Short   immotile  rods  • 

Ferment. 

ACIDI  LACTICI. 
ACTINOBACTER. 

Bacillus. 
Bacillus. 

aerobic. 
Short,  immotile  rods. 



AEROGENES 

Bacillus. 

capsule;  facul.  anse- 
rob. 

A  EROPHILUS 

Bacillus 

gle  and  in  pairs;  very 
resistant. 

AGILIS. 
ALBA. 
ALBA. 

ALBICANS  AMPLUS. 

Micrococcus. 
Beggiatoa. 
Sarcina. 
Micrococcus. 

immotile;            oval 
spores;  aerobic. 
Mobile  diplococci  with 
fine  flagella. 
Cocci  and  spirals  with 
sulphur. 

Small  cocci  in  packets. 
Large  cocci  and  diplo- 

Red pigment. 
White  pigment. 

ALBICANS  TARDIS- 

Micrococcus. 

cocci. 
Diplococci  colored  by 

IMUS. 

ALBICANS  TARDUS. 

Micrococcus. 

Gram. 
Diplococci  not  motile 

ALLII. 
AMYLIFERUM. 

Bacillus. 
Spirillum 

Very  small  rods. 
Rigid     spirilla     with 

Alkaloid  pigment 

spores  ;    turns    blue 
with  iodine. 

OF    THE    PRINCIPAL   BACTERIA. 

OF   THE   PRINCIPAL   BACTERIA. 

NON-PATHOGENIC  BACTERIA. 


239 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Not  liquefy  ;  membranous 
growth. 
Not  liquefy;  small  white 
points     porcelain  -  like  ; 
slow. 
Growth  faster  than  above  ; 
appearance  same. 

Produces      acetic-acid 
fermentation. 
Lactic-acid    fermenta- 
tion;       precipitates 
caseine. 
Alcohol  is  formed  after 
the   lactic-acid    fer- 
mentation. 
Causes      fermentation 

Air. 
Air  ;  sour  milk. 

Sour  milk. 
Air. 

Kiitzing. 
Pasteur. 

Grotenfeldt. 
Duclaux. 

Rapid  growth  ;  round,  con- 

with gas  and  alcohol. 

Digestive  tract. 

Miller. 

centrically-arranged  col- 
onies ;  not  liquefy. 

Old  cultures. 

Liborius. 

low-gray  colonies. 

Drinking-water. 

All  Cohens. 

a  cone  with  rose-red  color. 

Vauch 

Air  and  water 

colonies. 
Slowly  liquefy;  gray  col- 
onies;     growth     fairly 
rapid. 

Is  colored  by  Gram's 
method. 

Vaginal  secretion. 

Bumm. 

Bumni 

liquefying;     very    slow 
growth. 
Grows  slowly  on  surface 

Skin  in  eczema. 

Unna, 

the     boundary     raised  ; 
twice  as  large  as  above. 
Bright  green   pellicle   on 
agar. 

Decomposes  albumin. 

Green  slime  of 
onions. 
Water. 

TommasolL 
Griffths. 
VanTiegham, 

240 


CHIEF    CHARACTERISTICS 

NON-PATHOGEN  1C 


Name. 

Genus. 

Biology. 

Product. 

AMYLOBACTER. 
AQUATILIS. 

Bacillus. 
Micrococcus  . 

See  Butyricum,  with  wh 
Very  small  cocci  in  ir- 

ich it  is  identical. 

regular  groups. 

AKACHNOIDEA. 

Beggiatoa 

Very  thick   filaments 
containing  sulphur; 
motile. 



ARBORESCENS. 

ATTENTTATUM. 

Bacillus. 
Spirillum. 

Thin  rods,  with  round- 
ed ends  in  threads, 
and  singly;  iramotile. 

Threads  with  narrow- 

Yellow pigment. 

ed  ends. 

AURANTIACA. 

Sarcina. 

Small  cocci  in  pairs  and 
tetrads  ;         strongly 
aerobic. 

Orange-yellow  pig- 
ment. 

AURANTIACUS. 

Bacillus. 

Motile,     short     thick 
rods,  often  in   long 
threads. 

Orange-yellow  pig- 
ment. 

AURANTIACUS. 

Micrococcus. 

Oval  cocci  in  pairs  and 
singly  ;  immotile. 

Orange-yellow  pig- 
ment in  water,  al- 
cohol, and  ether; 
insoluble. 

AUREA. 

Sarcina. 

Cocci  in  packets. 

Golden-colored  pig- 
ment; soluble  in 
alcohol. 

AUREUS. 

Bacillus. 

Straight    motile    rods 
lying  parallel. 

Golden-yellow  pig- 
ment. 

BALTICUS. 

Bacillus. 

Short  rod. 

Phosphorescence. 

BIENSTOCKII. 

15  1  1  "L  HOT  HI  I 

Bacillus. 
Micrococcus 

See  Putrificus,  coli. 
Groups   of   cocci   sur- 

BRUNNEUS. 
BUTYRIC-ACID  FER- 
MENTATION. 

(ascococcus). 

Bacillus. 
Bacillus. 

rounded   with    cap- 
sule ;  zooglcea  aerobic. 
Motile  rods. 
Large,  slender  motile 
rods  in  pairs  ;  spores  ; 
facul.  anserobin. 

Brown  pigment. 
Diastase. 

OF    THE    PRINCIPAL    BACTERIA. 
BACTERIA.— CONTINUED. 


241 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Light-yellow  colonies  •  ser- 

Old distilled  water. 

Bolton. 

rated  edges. 

Sulphur  water 

Agardh. 

London  Water- 

Francland. 

oval  centre   like    roots; 
later  on  colored  yellow  ; 
slowly  liquefy. 

works. 
Stagnant  water. 

Warming. 

Rapidly     liquefy  •      little 

Air  and  water. 

Koch. 

orange-yellow    colonies, 
not  growing  in  high  tem- 
perature. 

Slowly  growing*  nail  cul- 

Water. 

Francland. 

tures;       shining       and 
orange-yellow;    not  liq- 
uefy. 
Round  orange-yellow  col- 

Water 

Cohn. 

onies,  mostly  on  surface  ; 
slow  growth  ;  not  lique- 
fying- 
Liquefv  ;     bright     golden 

Exudate  of  pneu- 

Mace. 

layer  on  potato. 
Slow-growing,  chrome-yel- 

monia. 
Water  and  skin  of 

Adametz  and 

low,  whetstone  in  shape; 
not  liquefy. 
Do    not   liquefy;    require 
glucose  for  growth. 

Creamv  layer  on   surface 

eczema. 
Baltic  Sea. 

Putrid  broth. 

Unna. 
Fischer. 

Cohn. 

of  gelatin. 

Maize. 

Schroter 

Liquefy  rapidly  ;  gray  veil 
on  surface  of  potato. 

Casein  ppt.  and  changec 
into  butyric  acid  ;  am- 
monia set  free. 

Air. 

Hueppe. 

16 


242 


CHIEF    CHARACTERISTICS 

NON-PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

BUTYRICUM    (amy- 
lobacter). 

Clostridlum. 

Thick  motile  rods  en- 
larging for  the  spores; 
obligat.  aerobic. 

Amyloid  substance. 

CCERULEUS. 

Bacillus. 

Rods  in  long  chains. 

Blue  pigment,  not 

soluble  in  water, 

alcohol,  or  acid. 

CANDICANS 

Micrococcus. 

Masses  of  cocci. 

(Candidas). 

CAROTARUM. 

Bacillus. 

Threads  of  rods  that 

bend  in  various  di- 

rections ;  oval  spores. 

CATENULA. 

Bacillus. 

Motile  rods  with  spores. 

CAUCASICUS. 

Bacillus. 

Motile  rods,  with  spores 

in  each  end. 

CERASINUS  siccus. 

Micrococcus. 

Very  small  cocci,  singly 

Cherry-red       pig- 

and in  pairs  ;  aerob. 

ment. 

CEREUS  ALBUS. 

Micrococcus. 

Cocci  in  short  chains 

and  bunches,  colored 

by  Gram. 

CEREUS  FLAVUS. 

Micrococcus. 

Staphvlo.  and  strepto., 

and'  in  zooglcea,  col- 

ored by  Gram. 

CHLORINUS. 

Bacillus. 

Large     rods,     motile, 

Green  pigment,  sol- 

green-colored, due  to 

uble  in  alcohol. 

chlorophyll;  aerobic. 

CHLORINUS. 

Micrococcus. 

Cocci  in  zoogloea. 

Green  pigment,  sol- 

uble  in    alcohol 

and  water. 

ClNNABAREUS. 

Micrococcus. 

Large    oval   cocci    in 
pairs;  aerobic. 

Brown  -  red       pig- 
ment ;  foul  odor. 

ClTREUS. 

Bacillus  (asco.). 

Straight  and  bent  rods 
in  bundles  ;  motile. 

Citron  yellow  pig- 
ment. 

ClTREUS. 

Micrococcus. 

Large  round  cocci  in 
chains  of  eight  and 

Cream-colored  pig- 
ment. 

more. 

ClTREUS    CONGLOM- 

Micrococcus. 

Diplococci  and  tetrads  * 

ERATUS. 

aerobic. 

CliAVIFORMIS. 

Bacillus 

Small  rods;  spores;  true 

(Tyrothrix). 

a  mt  robin. 

OF    THE    PRINCIPAL    BACTERIA. 
BACTERIA.— CONTINUED. 


243 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Not  cultivated. 
Liquefy  ;  a  deep-blue  layer 

Forms  butyric  acid  in 
presence     of    lactic 
acid. 

Air,  earth,  and  wa- 
ter. 

Water. 

Prazrnowski 
and  Van 
Tiegham. 
Smith. 

on  potato. 
Not  liquefy  ;  nail-shaped  in 

Air  around  old  cul- 

Flueee 

test-tube. 
Rapidly  liquefy  on  surface 

tures. 
Cooked  carrots  and 

A  Koch 

a  network  centre  on  po- 
tato; round,  light  gray; 
grow  rapidly. 

beets. 
Old  cheese 

ment.                fl 
TaT-manfe     milk,^^B 

R^efyr  *  grain. 

Kern 

ducing     the    kUPV 
drink. 

Water 

List 

ing  cherry-red  scum,  not 
developed  on  gelatin. 
Not   liquefy;   small  wax- 
like  drops;    thick   gray 
layer  on  potato  ;  growth 
rapid. 
Not  liquefy  •   dark-yellow 

...... 

Pus. 
Pus. 

Passet. 
Passet 

colonies;    wax-like    ap- 
pearance. 
Liquefy  *    greenish-yellow 

Water. 

Engelman. 

colonies. 
Yellow-green  layer  on  gel- 

Boiled eggs. 

Cohn. 

atin. 
Not  liquefy;  slow  growth* 

Air  and  water. 

FlQcrge 

bright-red  points. 
Slow    growth  •    after    two 

Skin  in  eczema 

TJnna  and 

weeks  small  yellow  points 
which  take  various  shapes 
on  potato;  citron  -yellow 
layer  ;       growth       more 
rapid. 
Dirty   cream  -colored    col- 

Water. 

Tommasoli. 
List. 

onies,  which  are  raised 
and  moist. 
Lemon-yellow  colonies 

Bumm 

Ferments  milk,  giving 

rhaglc  pus. 
Fermenting  albu- 

Duclaux. 

rise  to  alcohol. 

min. 

244 


CHIEF    CHARACTERISTICS 

NON-PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

CONCENTRICUM. 

CORONATUS. 
CORYZJE. 

CREPESCULUM. 

CYANEUS. 
CYANOGENUS  (blue 
milk). 

DlCHOTOMA. 
DlFFLUENS. 
DlSTORTUS. 

DYSODES. 

ENDOPARAGOGICUM. 
ERYTHROSPORUS. 

FIGURANS 
(mycoides). 

FlLIFORMIS. 
FlSCHERI. 
FlTZIANUS. 

Spirillum. 

Micrococcus. 
Micrococcus. 

Micrococcus. 

Micrococcus. 
Bacillus.      Jm 

'f' 
Cladothrix. 

Micrococcus. 

Bacillus 
(Tyrothrix). 
Bacillus. 

Spirillum. 
Bacillus. 

Bacillus. 

Bacillus 
(Tyrothrix). 
Bacillus. 

Bacillus. 

Thick    motile    spirals 
withflagella;  aerobic. 

Cocci  singly  and  strepto- 
cocci; aerobic. 
Large  diplococci  with 
rounded    ends,    the 
contact  surfaces  flat. 

Round  and  oval  cocci, 
singly  and  in  zooglcea. 
.Oval  cells. 
%totile  rods  in  chains  ; 
^spores  ;  aerobic. 

Various     forms  —  rods, 
spirals,  and  cocci,  in 
long  threads. 
Oval  cocci  ;  aerobic. 

Motile   rods  ;    spores  ; 
aerobic. 
Long  and  short  rods; 
spores. 

Dry  motile  spirals,  join- 
ed in  peculiar  shapes. 
Motile       rods        and 
threads  ;  spores,  slen- 
der. 
Large      motile     rods; 
spores;  long  threads; 
aerobic. 

Short     motile     rods  ; 
spores  in  one  end. 



Blue  pigment. 
Alkali  and  a  pig- 
ment   deepened 
by  acids. 

Fluorescent     pig- 
ment, soluble  in 
water. 
Alkali. 

An  odor  resembling 
peppermint  and 
turpentine. 

Greenish  -  yellow 
pigment. 

Phosphorescence, 

Short  rods  in  threads  ; 
spores  as  large  as  the 
rods. 

OF    THE    PRINCIPAL    BACTERIA. 
B  ACTEEIA  .—CONTINUED. 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Not  liquefying  •  concentri- 

Putrefying blood. 

Kitasato 

cally-disposed    colonies; 
very  slow   growth  ;   not 
growing  on  potato. 

Air 

Fliieee 

colonies. 
White,  raised  glassy   col- 
onies, at  first  like  pneu- 
uiococci,    later    culture 
flattened  ;  not  liquefying. 

No  pathogenic  action. 

Acute   coryzal  se- 
cretion. 

Putrefying     infu- 

Hajek. 
Cohn. 

Bluish-green  colonies 

sions. 
Cooked  potatoes 

Colm 

Not  liquefying  ;  small  white 
colonies. 

Cultivated  in  infusion   of 

Changes  milk  to  deep- 
blue  color. 

Air  of  certain  coun- 
tries. 

Water 

Fuchs. 
Cohn. 

plants. 
Do  not  liquefy  ;  small  gran- 

Air. 

Schroter. 

ular,    yellow,    colonies  ; 
green  fluorescence. 

Milk  made  viscid  and 

Air. 

Duclaux. 

casein  precipitated. 

Bread  and  yeast 

Zopf 

Trunk    of    worm- 

Sorokin. 

eaten  tree. 

Cohn 

fluorescence;  white  col- 
onies. 
Liquefying  •  root-like  pro- 

substances. 
Garden-earth. 

Flugate. 

cesses  extending  in  the 
gelatin  ;  feather  form  in 
test-tube. 

Causes  casein  to  be  pre- 

Duclaux. 

Not   liquefying*    requires 

cipitated  from  milk. 

Beyerinck. 

peptone  for  growth. 
Transparent   on    surface  ; 
dark  centre  in  the  deep  ; 
not  liquefying. 

Produces  ethylic  alco- 
hol in  meat  extract. 

Unboiled  hay-infu- 
sion. 

Zopf. 

246 


CHIEF    CHARACTERISTICS 


NON-PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

FLAVA. 
FLAVUS. 

Sarcina. 
Bacillus. 

Small  cocci  in  packets. 
Small  rods  ;  immotile. 

Pigment. 
Pigment. 

FLAVUS  DESIDENS. 

Streptococcus. 

Cocci  and  diplococci  in 
chains;  aerobic. 

Yellow-brown  pig- 
ment. 

FLAVUS    LIQUEFA- 

CIENS. 

FLAVUS        TARDI- 

GRADUS. 

FLUORESCENS   FCE- 

TIDUS. 

Micrococcus. 
Micrococcus. 
Micrococcus. 

Cocci  and  diplococci  in 
zooglcea. 
Cocci  in  short  chains, 
and  diplococci. 
Small  diplococci. 

Pigment. 

Chrome-yellow  pig- 
ment. 
Blue-green       pig- 
ment :  acids  turn 
red. 

FLUORESCENS 
LIQUEFACIENS. 
FLUORESCENS  NIVA- 

LIS. 

Bacillus. 
Bacillus. 

Short  motile  rods  ;  very 
thin. 
Short  rods  ;  motile. 

Green    fluorescent 
pigment. 
Blue-green       pig- 
ment. 

FLUORESCENS    PU- 

TRIDUS. 
FOERSTERI. 

Bacillus. 
Cladothrix. 

Motile  rods  ;  short,with 
rounded  ends. 

Threads     twisted     in 

Green   fluorescent 
pigment. 

spirals;   very  irreg- 
ular. 

FCETIDUM. 

Clostridium. 

Rods  of  varying  length  ; 
very  motile;  a  large 
spore    in    one    end; 
anaerobic. 

Strong  gas-produc- 
tion ;   very   foul 
odor. 

F<ETIDUS. 

FUSCESCENS. 
FULVUS. 

Micrococcus. 
Sarcina. 
Micrococcus. 

See  Orepesculum,  with 
Round  cocci. 

which  it  is  identi 

FUSCUS  LIMBATUS. 
FUSIFORME. 

Bacillus. 
Bacillus. 

Short  rods;   very  mo- 
tile ;       facultatively 
anaerobic. 
Spindle-shaped,     with 

Brown  pigment. 

GENICULATUS. 

GlGANTEUS        URE- 

Bacillus 
(Tyrothrix). 
Micrococcus. 

pointed  ends. 
Rods  variable  length; 
spores. 
Streptococci   in   thick 

A  bitter  substance. 

THRA. 

knots. 

OF  THE    PRINCIPAL    BACTERIA. 
BACTERIA.— CONTINUED. 


247 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Vomited  matter      ] 

Drinking-water 

Mace. 

colonies  ;  foul  odor. 

Fliiece 

onies. 

tures;  water. 
Air   and  old   cul- 

Flueee 

colonies. 
Softens     gelatin;     yellow 

tures;  water. 
Air. 

Flugge. 

beads,  isolated. 

Post-nasal  space 

Xlamann. 

that  later  on  sink  in,  sur- 
rounded by  violet-green 
color;  liquefying;  growth 
rapid. 
Liquefying  ;  white,  sunken, 

Water  and  air  ;  con- 

Flugge. 

iridescent  colonies. 
Quickly  liquefying;  growth 
rapid;  small  white  points; 
later  on,  surrounded  by 
blue-green  fluorescence. 
Not  liquefying;  transparent 

Colors  the  glacial  wa- 
ters green. 

junctival  sac. 
In  snow  and  ice  of 
Norway. 

All  putrefactions. 

Schmolck. 
Flugge. 

at  first,  then  green  flu- 
orescence  and    urinary 
odor. 

Lachrymal  canal. 

Cohn. 

Liquefying  ;  growth  rapid  ; 

Old  cheese  and  se- 

Liborius. 

small  colonies  that  soon 
become    filled    up    with 
fluid  and  assume  a  spher- 
ical form, 
cal. 

Conical  rusty-red  colonies. 

' 

rum  of  mice  in- 
oculated      with 
garden-earth. 

Excrement  of  horse. 

Cohn. 

Small       brown      colonies, 

In  foul  eggs. 

Scheiben- 

along  needle-track  little 
branches;  not  liquefy. 

Spongy    layer    on 

zuber. 
Warning 

sea-water. 

No  growth  on  gelatin  ;  on 

Normal  urine  and 

Lustgarten 

agar,  thin  drops;  nearly 
transparent;    very  slow 
growth;    in    bouillon,  a 
flaky  precipitate. 

urethra. 

248 


CHIEF    CHARACTERISTICS 

NON-PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

GRAVEOLENS. 

Bacillus. 

Small  rods,  nearly  as 

Foul  gas. 

broad    as    they    are 

long. 

H^EMATODES. 

Micrococcus. 

Cocci  in  little  zoogloea. 

Red  pigment. 

HANSENII. 

Bacillus. 

Medium  large  rods. 

Yellow     pigment  ; 

insoluble. 

HYACINTHI. 

Bacillus. 

Short   rods   in   dumb- 

bell shapes. 

HYALINA. 

Sarcina. 

Round  cocci  in  groups 



of  4  to  24. 

IANTHINUS. 

Bacillus. 

See  Bacillus  violaceus. 

INDICUS. 

Bacillus. 

Short  motile  rods;  no 
spores  ;        anserobin 

Scarlet  pigment  al- 
tered by  heat. 

facul. 

INTESTINAL  is. 

Sarcina. 

Very  regular    packets 
of  cocci,  eight  in  each. 



JEQUIRITY. 

Bacillus. 

Medium-sized       rods; 

Ferment        called 

spores. 

abrin. 

KtJHNIANA. 

Crenothrix. 

Long  threads,  breaking 

up  into  cocci.    They 

are  ensheathed. 

LACTEUS  FAVIFOR- 

MIS. 

Micrococcus. 

Diplococci;  not  decol- 
orized by  Gram. 

LACTIS  ERYTHROG- 

Bacillus. 

Short  immotile    rods; 

Yellow       pigment 

ENES. 

round  ends. 

and  red  pigment. 

LEPTOMITIFORMIS. 

Beggiatoa. 

Filaments        medium 

size. 

LEUCOMEL^ENUM. 

Spirillum. 

Two  or  three  spirals; 

dark  granular    con- 

tents ;    clear   spaces 

between. 

LlNEOLA. 

Bacillus. 

Short  motile    rods  in 

zooglo?a,with  flagella. 

LlODERMOS. 

Bacillus. 

Short     motile      rods  ; 

rounded  ends. 

OF    THE    PRINCIPAL   BACTERIA. 
BACTERIA.— CONTINUED. 


249 


Culture  Characters. 


Actions. 


Habitat. 


Discoverer. 


/ing ;  irregular  gray- 
ish, later  greenish,  colo- 
nies, with  very  foul  odor. 

Grows  best  on  white  of 
egg  at  37°  C. ;  red  layer. 

On  potato,  a  yellow  growth 
which  changes  with  age. 


Liquefying ;  oval  colonies ; 
scarlet-colored. 


Coloniesbrick-colored  from 
oxide  of  iron. 

Not  liquefying;  white  col- 
onies ;  grow  well  on  po- 
tato. 

Small,  round  yellow  dots, 
later  on  cup-shaped,  with 
rose-colored  periphery ; 
liquefying. 


Slimy  layer  on  potatoes. 

Liquefying;  transparent, 
then  thick  layer  on  po- 
tato; like  gum. 


Ferment   causes  oph- 
thalmia. 


Skin  between  toes. 


Sweat  of  man. 

Yellow  skin  of 
nutrient  infu- 
sions. 

Slime  of  diseased 

hyacinth-bulbs. 
Marshes. 


Intestine  of  mon- 
key. 

Intestine  of  fowls. 

Infusion  of  jequir- 

ity  bean. 
Drinking-water  of 

wells. 

Mucus    of   vagina 
and  uterus. 

In  red   milk    and 
faeces. 


Sulphur  waters. 

Water  over  rotting 
plants. 


Stagnant  water. 
Air  and  potatoes. 


Bordoni- 
Uffreduzzi. 

Zopf. 

Rasmussen. 

Wakker. 
Kiitzing. 

Koch. 

Zopf. 

Sattler. 

Rabenhorst. 

Bumm. 


Hueppe    and 
Grotenfeldt. 


Trfivisan. 
Perty. 

Muller. 
Fliigge. 


250 


CHIEF    CHARACTERISTICS 

NON-PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

LITORAUS. 
LlTOREUS. 
LlVIDUS. 

Merismopedia. 
Bacillus. 
Bacillus. 

Cocci  in  groups  of  fours, 
containing  sulphur. 
Oval    rods,    never    in 
chains  or  zooglcea. 
Medium-sized       rods; 
motile. 

Deep  blue-black 
pigment. 

LUTEA. 

Sarcina. 

Cocci  singly  and  in 
fours. 

Pigment  citron- 
yellow. 

LUTKUS. 
LUTKUS. 

Bacillus. 
Micrococcus. 

Short  immotile  rods, 
with  large  oval  spores. 

Oval  cocci. 

Pigment  ;    soluble 
in  water;    acids 
intensify. 
Pigment,  not  acted 
upon  by  acid  or 
alkali. 

LUTEUS. 

Micrococcus. 

Diplococci  very  motile. 

Yellow  pigment, 
turning  brown- 
red. 

MAIDIS. 

Bacillus. 

Rods  with  pointed 
ends  ;  very  motile  ; 
seldom  in  threads; 
oval  spores. 



MARSH. 

Spirillum. 

See  Plicatile. 

\  MEGATERIUM. 

Bacillus. 

Large  motile  rods; 
spores;  aerobic. 



MELANOSPORUS. 

Bacillus. 

Rods;  aerobic. 

Black  pigment,  not 
acted  upon  by 
acids  or  alkalies. 

MERISMO-PEDI- 

OIDES. 

Bacillus. 

Threads  of  rods  which 
are  formed  from 
cocci-like  spores  ; 
zooglcea  in  packets. 

MESENTERICUS  FUS- 
cus  (potato). 

Bacillus. 

Small  motile  rods  with 
spores. 



MESENTERICUS  VUL- 
OATUS  (potato). 

Bacillus. 

Thick  motile  rods  in 
threads;  spores. 

Diastase. 

OP    THE    PRINCIPAL   BACTERIA. 
BACTERIA.— CONTINUED. 


251 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Sea-water. 

Oersted    and 

Sea-water 

Rabenhorst. 
Warming 

Ink-spot    at    first,    slowly 
liquefying;      blue-violet 
colored   later   on  ;   slow 
growth. 
Not  liquefy  ing-  little  eleva- 

...... 

Berlin  Water- 
works. 

Air               •  • 

Plogge      and 
Proskauer. 

Schroter. 

tions  ;  citron-yellow  cen- 
tre ;  yellow  layer  on  po- 
tato. 
Not  liquefying  ;   irregular 

Air 

Fliigge. 

in   form  ;   golden-yellow 
colored. 
Do  not  liquefying  •   small 

Air 

Schroter. 

citron-yellow  colonies  on 
potato. 
Round,    light-yellow    col- 
onies, growing  larger  in 
a  few  days  ;  on  potato  a 
slimy      covering      with 
mouldy  odor  ;  slowly  liq- 
uefying. 

Gray  points  in  deep,  veil- 
like  on  surface  ;  liquefy- 
ing; on  potato,  a  wrink- 
led   skin    of    brownish 
color. 

In  solutions  of  sugar 
an  aldehyde  pro- 
duced. 

Water. 

In   maize   and  in 
pellegra;  faeces. 

Adametz. 

Paltauf    and 
Heider. 

De  Bary 

thick  layer  on  potato. 
First  gray  then  black  pel- 

Air and  potatoes 

Eidam 

licle. 

Zopf 

Liquefying;    white    colo- 
nies, ray-like  periphery  ; 
brown  layer  on  potato. 

bellow  colonies,  dark  cen- 
tre, ciliary  processes  at 
periphery  ;  brown  layer 
on    potato,   penetrating 
the  substance. 

Coagulates  milk  and 
forms  diastase  out  of 
starch. 

Potato. 

Air  and  old  pota- 
toes. 

Flugge. 
Fliigge. 

252 


CHIEF   CHARACTERISTICS 

NON-PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

MESENTEROI  DBS. 

LeuconostoCt 

Masses  of  cartilaginous 

MILLER'S. 

Bacillus. 

zooglcea,  composed  of 
rods  and  cocci;  ar- 
throspores. 

Delicate  rods  slightly 

MINUTA. 

Sarcina. 

curved;  im  motile. 
Cube-shaped  packets. 

MlKA  BILLS. 

Beggiatoa. 

Very     wide     threads 

MULTI  PEDIC  ULOSUS. 

Bacillus 

rounded    ends    and 
curled;  sulphur  gran- 
ules. 

Long  slender  rods 

MULTISEPTATA. 

Phragmidio- 

NASALIS. 

thrix. 
Micrococcus. 

ing  cocci  which  are 
not  free;  they  have 
no  sulphur,  and  are 
not   enclosed    in    a 
sheath. 

Diplococci,  motile  ;  also 

NAVICULA. 

NlTRIFICANS. 
NlVEA. 

Bacillus. 

Micrococcus. 
Beggiatoa. 

streptococci. 
Spindle-shaped  rods. 

Small  cocci. 
Very  thin  filaments. 

Amyloid  material 
Forms  saltpetre. 

NODOSUS  PARVUS. 

Bacillus 

Rods  formed  at  angles  • 

OBLONGUS. 

Micrococcus 

immotile. 
Motilecocci  singly  and 

OCHROLEUCUS. 
PALUDOSA. 

Micrococcus." 
Sarciua. 

in  filaments  ;  aerobic. 

Cocci  in  pairs  and  pack- 
ets; spores. 

Spherical,  transparent, 

Yellow  pigment. 

colorless  coccL 

OF    THE    PRINCIPAL    BACTERIA. 
BACTERIA  .—CONTINUED. 


253 


Culture  Characters. 


Actions. 


Habitat. 


Discoverer. 


Converts  molasses  into 
a  gelatinous  mass. 


Liquefies ;  not  growing  on 
the  surface. 

Grows    slowly;    reacts   to 
iodine,  turning  blue. 


Insect-shaped  colonies. 


Grayish  points,  raised, 
opaque ;  rapid  growth ; 
not  liquefying. 


White  flakes. 

Slow  growth  at  37°  C.;  in 
agar  a  white  line,  which 
in  the  centre  becomes 
porous. 

Grows  best  in  cultures  to 
which  glucose  and  am- 
mon.  tartrate  have  been 
added. 

Liquefying;  slow  growth; 
thin  yellow  membrane; 
sulphurous  odor. 


Causes    gluconic    fer- 
mentation. 


Beet- root  juice. 

Caries  of  teeth. 
Sour  milk. 
Sea-water. 


Potatoes. 
Sea-water. 


Nasal  space  and  se- 
cretion. 

Potatoes. 

Soil. 

Sulphur  waters. 

Urethral  secretion. 


Beer. 


Urine. 


Water  from  sugar- 
iactory. 


Cienkowski. 


Miller. 
De  Bary. 
Cohn. 


Fliigge 


Hack. 

Reinke     and 
Berthold. 

VanTiegham 
Rabenhorst. 
Lustgarten. 


Boutroux. 

Prove. 
Schroter. 


254 


CHIEF    CHARACTERISTICS 

NON-PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

PASTEURIANUS 

Bacillus 

Differs  from  bacil  aceti 

PFLUGERI. 
PHOSPHORESCENS 

GELIDUS. 

PHOSPHORESCENS 

INDICUS. 

PHOSPHORESCENS, 
North  Sea. 

PHOTOMETRICUS. 
PLICATILE. 

Bacillus. 
Bacillus. 
Bacillus. 
Bacillus. 

Bacillus. 
Spirillum. 

in  that  the  cells  con- 
tain an  amyloid  mat- 
ter. 
Short  rods  in  threads. 

Motile;    round,   short 
rods;  aerobic. 
Large  motile  rods. 

Motile  rods. 

Motile,        red-colored 
rods. 

Long      motile,      thin 

Phosphorescence. 
Phosphorescence. 
Phosphorescence. 
Phosphorescence. 

Sulphur   and   red 
pigment   caused 
by  light. 

POLYMYXA. 
PRODIGIOSUS. 

PROTEUS  MIRABILIS. 
PROTEUS    VUL- 

Clostridium. 
Bacillus. 

Bacillus. 
Bacillus. 

spirals;  round  ends. 
Motile  rods  in  threads 
with  spores. 
Short     motile      rods; 
aerobic. 

Very  motile,  short  rods; 
aerobic. 

Rods  sometimescurved, 

Amyloid,     colored 
blue  by  iodine. 
Red  pigment,  sol- 
uble  in    alcohol 
trimethylauiine. 

GARIS. 

as  spirillum. 
Motile  rods 

PSEUDO-DIPHTHE- 

Bacillus. 

Small  rods   similar  to 

PUTRIFICUS  COLI. 
PYOGENES  TENUIS 

Bacillus. 
Micrococcus 

the  true  bacillus  ;  im- 
motile. 

Slender   motile    rods  ; 
long  threads  ;  spores. 



RADIATUS. 

' 

RADIATUS. 

Bacillus. 

: 

Streptococcus 

Motile  rods  with  round- 
ed ends;   anaerobic; 
oval  spores. 

Small  cocci  in  chains 

Strong  -   smelling 
gas. 

OP    THE    PRINCIPAL    BACTERIA 
BACTERIA.— CONTINUED. 


255 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Heavy  beers. 

Putrid   meat   and 
fish. 
Salt  fish. 

Tropical  seas. 
Water  around  Kiel. 

Hansen. 

Ludwig. 
Forster. 
Fischer. 
Fischer. 

Engelman. 

Ehrenberg. 
Prazrnowski. 
Ehrenberg. 

Hauser. 

Hauser. 
Hauser. 
Wellenhof. 

Bienstock. 

Rosenbach. 
Luderitz. 

Fltigge. 

Not  liquefg;  requires  glu- 
cose ;  grows  well  on  potato. 
Not  liquefying  ;  grows  best 
with  glucose  and  salt. 
Liquefying  ;  grows  best  at 
30°  C. 
Liquefying;  colonies  look 
as  if  punched  out  ;  grows 
best  at  15°  C. 
Movements  depend  upon 
light. 

• 

Causes  fermentation  in 
dextrin  solutions. 

Stagnant  water. 

Thick  skin  on  potato. 

Little  red  colonies;  lique- 
fying rapidly  ;  especially 
abundant  on  potatoes. 
Liquefying  slowly  ;  opaque 
centre,    irregular     pro- 
cesses. 
Liquefying  quickly. 

Not  liquefying;  thick  white 
layer  on  potato. 
Grows    at    ordinary    tem- 
perature,  rapidly   form- 
ing on  surface  a  brown- 
ish    growth  ;     pin-head 
colonies  raised  above  sur- 
face; not  liquefying. 

Bread    and    pota- 
toes. 

Putrefaction. 

Putrefaction. 
Putrefaction. 

In        diphtheritic 
membrane     and 
normal  pharynx. 

Human  faeces. 

Closed  abscesses. 
In  serum  of  white 
mice   inoculated 
with  earth. 

Air. 

Not  virulent. 
Decomposes  albumen. 

On  agar,  a  glassy  growth. 
Liquefying  ;  growth  rapid  ; 
colonies     like     moulds, 
from     centre     radiating 
in    all    directions     and 
through  the  gelatin  ;  the 
air  must  be  excluded. 
Liquefying;  white  colonies 
with  greenish  tinge  ;  fun- 
nel-shaped in  test-tube. 

Not  pathogenic. 

256 


CHIEF    CHARACTERISTICS 

NON-PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

KAMOSUS  LIQUEFA- 

Bacillus. 

Motile  rods. 

CIKNS. 

REITENBACHII. 

Merismopedia. 

Cocci    in    packets    or 

plates  ;  colorless  cell- 

wall  containing  chlo- 

rophyll. 

ROSACEUS. 

Micrococcus. 

Large  cocci  in  pairs  and 

Red  pigment. 

tetrads. 

ROSEA. 

Sarcina. 

Spherical  cocci  in  cubi- 

cal packets. 

ROSEA  PERSEINA. 

Beggiatoa. 

Long  rods  with  cocci- 

Pigment  called  bac- 

shaped     bodies      in 
them,  containing  sul- 

terio-purpurin. 

phur  and  a  red  pig- 

ment. 

ROSEUM. 

Spirillum. 

Very  short  curved  rods  ; 
motile  and  spores. 

Pigment  soluble  in 
alcohol. 

RUBEK. 

Bacillus. 

Motile  rods  in  groups. 

Brick-red  pigment. 

RUBRCM. 

Spirillum. 

Motile  ;  short  spirilla  ; 
aerobic. 

Pale-rose  pigment. 

RUKUM. 

Spirillum. 

Long  motile  spirals. 

Red-rose  pigment. 

RUGUI/A. 

Spirillum 

Motile    rods,    in    long 

(vibrio). 

spirals,  singly  and  in 
chains,  with  flagella 

and  spores  ;  anaerobic. 

SAPROGENES. 

Bacillus. 

Large    rods,    terminal 

spores;  facultatively 

anaerobic. 

SCABER. 

Bacillus 

Short   motile  rods  in 

Tyrosin  and  leucin 

(Tyrothrix). 

chains;  spores;  aerobic. 

,are  formed. 

SCHEURLEN'S. 

Bacillus. 

Short     motile      rods  ; 

spores. 

SEPTICUS. 

Bacillus. 

Non-motile     rods     in 

threads  and  spores; 

anaerobic. 

SERPENS. 

Spirillum. 

Long,   lively   threads, 



with  three  windings. 

OF    THE    PRINCIPAL    BACTERIA 
BACTEKIA.— CONTINUED. 


257 


Culture  Characters. 


Actions. 


Habitat. 


Discoverer. 


Liquefying;  concentrical 
colonies ;  funnel-shaped 
in  test-tube. 


Not  liquefying;  small  red 
knobs,  with  faecal  odor. 


Not  liquefying;  thick  vio- 
let colonies ;  deep  red  on 
potato. 


Not  liquefying ;  grows  slow- 
ly ;  pale-rose  colonies. 


Air. 


Liquefyin 

(V    ( 

odor. 


;  round 


. 

yellow  dots  with  zone; 
fecal 


Causes  cellulose  to  fer- 
ment. 


Grows  slowly ;  foul  odor. 


Growth  best  at  39°  C.; 
slowly  liquefying  on  po- 
tato; a  yellow,  wrinkled 
skin,  underneath  which 
a  red  color. 


Air. 

Marshes. 
Marshes. 

Blennorrhagic  pus. 

Boiled  rice. 
Dead  mice. 

Stagnant  water. 

Vegetable  infu- 
sions and  tartar 
of  teeth. 

Putrefaction. 


In  carcinomatous 
and  normal  mam- 
ma. 


Putrid  blood. 


Stagnant  water. 


Flugge. 
Caspary. 

Flugge. 

Schroter. 

Zopfc 

Mace. 


Frank. 

Esmarch. 

Perty. 

Miiller. 


Rosenbach. 

Duclaux. 
Scheurlen. 

Klein. 
Muller. 


17 


258 


CHIEF    CHARACTERISTICS 

NON-PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

SIMILIS. 

SPINOSUS. 

SUBFLAVUS. 
SUBTILIFORMIS. 

SUBTILIS  (hay   ba- 
cillus). 

SYNCYANEUS. 
SYNXANTHUS   (yel- 
low milk). 

TENUE. 
TENUIS. 

TERMO. 

TREMULUS. 

TUMESCENS. 
TURGIDUS. 

ULNA. 

UNDULA. 
UREJE. 

URINJE. 

Bacillus. 
Bacillus. 

Micrococcus. 
Bacillus. 
Bacillus. 

Bacillus. 
Bacillus. 

Spirillum. 

Bacillus 
(Tyrothrix). 

Bacillus. 

Bacillus. 

Bacillus. 

Bacillus 
(Tyrothrix). 

Bacillus. 

Spirillum. 
Bacillus. 

Sarcina. 

Immotile  rods;  trans- 
parent spores. 
Large     motile     rods  ; 
spores;   true  an  aero- 
bin. 
Diplococci   colored  by 
Gram's  fluid. 

Immotile       rods      in 
threads  ;  transparent 
spores. 
Large  motile  rods,  three 
times    longer    than 
broad,    in     threads, 
with     flagella     and 
spores;  aerobic. 
Same  as  Cyanogenus. 
Short,  thin  motile  rods. 

Large    motile    spirals 
with  flagella. 
Motile    rods    in    long 
chains;  spores. 

Short  motile,  cocci-like 
rods  in  zooglrea. 

Motile  rods  with  flagel- 
la and  large  round 
spores. 
Short  rods  with  spores. 

Short  immotile  rods  in 
long  chains  ;  spores  ; 
aBrobic. 
Very    large    rods     in 
chains    and    singly  ; 
not  very  motile;  large 
spores. 
Long     motile    spirals, 
with  flagella. 
Short     rods  ;     spores  ; 
aerobic. 

Small  cocci  in  families. 

Yellow  pigment, 
soluble  in  water; 
similar  to  aniline 
colors. 

Carbonate  of  am- 
monium. 

Ferment,  propyla- 
mine. 

OF    THE    PRINCIPAL    BACTERIA. 
BACTERIA.— CONTINUED. 


259 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Grows  rapidly. 

Liquefying;  spiny  periph- 
ery ;    foul  odor  due   to 
methyluiercaptan. 
Liquefying;  yellow  dots. 

Grows  best  at  37°  C. 

Liquefying;    gray  centre, 
wreath-like  border  ;  thick 
layer  on  potato. 

In   boiled   milk  a  yellow 
pigment  is  formed. 

Albuminous  decompo- 
sition. 

Human  faeces. 
Garden-earth. 

Vaginal    secretion 
and  lochial  dis- 
charges. 
Human  faeces. 

Soil  and  dust,  hay, 
etc. 

Boiled    milk    and 
potatoes. 

Stagnant  water. 

Fermenting  cheese 
and  milk. 

Connected       with 
putrefaction     of 
plants. 

Putrefying  plants. 

Boiled  carrots. 

Fermenting    milk 
and  cheese. 

Putrefying    water 
and  boiled  eggs. 

Vegetable  infusions. 
Stale  urine. 

Bladder. 

Bien  stock. 
Luderitz. 

Bumm. 
Bienstock. 
Ehrenberg. 

Ehrenberg. 

Ehrenberg. 
Duclaux. 

Dujardin. 

Zopf. 
Duclaux. 

Cohn. 

Muller. 
Miquel. 

Welcker. 



Precipitates  casein  ; 
forms  a  pellicle  on 
milk. 

Liquefying;   opaque   cen- 
tre, yellow  layer   next, 
and  the  periphery  lobed  ; 
funnel-shaped    in    test- 
tube. 

On  boiled  carrots  a  wrin- 
kled gelatinous  disk. 
A  pellicle  formed  on  sur- 
face of   milk;    a  heavy 
precipitate  beneath. 
On  bofled  egg  little  zoog- 
Io2a. 

Resembling   a   globule  of 
fat  ;  grows  well  in  mu- 
cous urine. 

Splits  urea  into  am- 
monii  carbonas. 

260 


CHIEF    CHARACTERISTICS 

NON-PATHOGENIC 


tfarae. 

Genus. 

Biology. 

Product. 

UROCEPHALUS. 

Bacillus 

Cylindrical  motile  rods 

VENTRICULA. 

(Tyrothrix). 
Sarcina. 

with  spores;  anaero- 
bic. 
Cubical  packets  of  8  to 

VENTRICULI. 

Bacillus. 

64  cocci. 
Rods  motile   often  in 

VERSICOLOR. 

Micrococcus. 

bundles  of  four. 
Small  cocci. 

VlOLACEUS. 
VlOLACEUS. 
VlRENS. 
VlRESCENS. 

VlRGULA. 

Bacillus. 
Bacillus. 
Bacillus. 
Bacillus. 

Bacillus 

Motile  rods,  round  end  ; 
spores. 

Immotile  rods,  forming 
large  spores. 

Straight  rods;  spores; 
iminotile;         green 
tinged. 
Short  motile  rods  with 
flagella  very  brdad. 

Slender  immotile  rods  • 

Violet  pigment,  sol- 
uble in  alcohol. 

Violet       pigment, 
like  aniline. 

Supposed   to   con- 
tain chlorophyll. 

Deep-green       pig- 
ment,     turning 
yellow-brown. 

VlRIDIS 

(Tyrothrix). 
Bacillus 

spores  aerobic. 
Little  immotile  rods  • 

Viscosus. 
Viscosus. 

VlTICULOSUS. 

Bacillus. 

Micrococcus. 
Micrococcus. 
Spirillum 

oval  spore,  which  is 
tinged  green. 
Motile   rods,   rounded 
ends,  usually  in  pairs. 

Streptococci  of  globular 
cells. 

Oval    cocci    in    large 
groups. 

Green  pigment. 

Gummy  substance, 
called  viscosa,  and 

ferment. 

ZOPFII. 

Bacillus. 

flagella. 
Long  motile  rods  break- 

ing  up   into  spores 
like  cocci. 

OF   THE    PRINCIPAL    BACTERIA. 
BACTEKIA.— CONTINUED. 


Culture  Characters. 


Actions. 


Habitat. 


Discoverer. 


Not  liquefying. 

Round  colonies  with  dark 

centre ;  slow  growth ;  not 

liquefying. 
Not  liquefying ;  iridescent 

yellow  surface. 
Not  liquefying ;  centre  deep 

violet ;  color  remains  on 

agar  a  long  time. 
Liquefying ;     transparent 

colonies,  surrounded  by 

violet  zone. 


Deep  round  colonies,  the 
vicinity  colored  green; 
grows  on  surface;  slow 
growth ;  not  liquefying. 


Rapid  growth,  liquefying; 
small  hair-like  processes 
from  colonies;  later  on, 
viscid  and  in  threads, 
with  green  fluorescence. 


Not  liquefying;  a  fine  net- 
work in  the  colony ;  mu- 
coid  layer  on  potato. 


Peptonizes  albumen. 


Mucoid    fermentation 
in  wine  and  beer. 


Not  liquefying;  forms  thick 
coils  like  braided  hair. 


Fermenting  milk. 


Contents  of  stom- 
ach. 

Stomach  of  dogs 
fed  on  meat. 

Air. 
Water. 


Boiled  potato  and 
water. 


Stagnant  water. 
Green  sputum. 

Milk. 
Water. 

Water  and  earth. 

Beer  and  wine. 
Air. 

Marshes. 

Intestinal  contents 
of  fowls. 


Duclaux. 

Goodsir. 
Raczynssky,, 

Fliigge. 
Zopf. 

Schroter. 

VanTiegham. 

Frick. 

Duclaux. 
VanTiegham. 

Franc!  and. 

Pasteur. 
Flugge. 

Ehrenberg. 
Kurth. 


262 


CHIEF    CHARACTERISTICS 


PART  II. 


Name. 

Genus. 

Biology. 

Product. 

AEROGENES  CAPSU- 

Bacillus. 

Usually  found  in  pairs, 

Gas   with   charac- 

LATUS. 

resembling       diplo- 

teristic  odor. 

cocci  ;       capsulated  ; 

obligate  anaerobe. 

ALVEI. 

Bacillus. 

Eods  with  large  spores. 

AMYLOVORUS. 

Micrococcus. 

Oval    cells,    never    in 

Forms         butyric 

chains. 

acid. 

A  ANTHRACIS   SYMP- 

Bacillus. 

Large     slender     rods 

Rancid  odor. 

TOMATICI. 

with     swellings     at 

spore  ;  anaerobic. 

.    ANTHRAX. 

Bacillus. 

Straight  rods,  slightly 

Toxalbumin. 

t 

concave  ends  ;  immo- 

tile;  aerobic;  spores. 

ARTICULORUM 

Micrococcus. 

Oval     cocci     in    long 

(diphtheriticus). 

chains,  identical  with 

pyogenes. 

BOMBYCIS. 

Micrococcus. 

Oval    cocci   in   chains 

and  zoogloea;  motile. 

BOTULINUS. 

Bacillus. 

Large  rounded   ends  ; 

Butyric  acid;  and 

motile;     flagellated; 

a  powerful  toxin. 

anaerobe. 

BUBONIC  PLAGUE. 

Bacillus. 

Short  thick  rods  with 

indistinct  capsule. 

BUCCALIS. 

Leptothrix. 

Long  threads  in  thick 

bundles,   containing 

masses  of  cocci  and 

spirals. 

CATTLE      PLAGUE 

.          ,    _    . 

(Texas  fever). 

^ 

wine 

CAVICIDA. 

Bacillus. 

Little  rods  twice  as  long 
as  broad. 

Propionic         acid 
through   decom- 

CHAUV^I     (symp- 
tomatic anthrax), 
(Rauschbrand). 

Bacillus. 

Large  rods  with  a  spore 
at  one  end,  assuming 
the  clostridium  type  ; 

position  of  sugar. 
Toxalbumin. 

motile;     never*   in 

threads  ;  true  anaero- 

l 

bin. 

OF    THE    PRINCIPAL    BACTERIA. 


263 


PATHOGENIC  BACTERIA. 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Acid    reaction    in    litmus 

Causes    fermentation  ; 

Intestinal        con- 

Welch. 

milk  ;  coagulates  casein 
with      cavity  -formation 

can  produce  gas  from 
proteid  alone. 

tents  ;        earth  ; 
water:           raw 

due  to  gas. 

foods. 

Liquefying;    growths    ra- 

Produces a  disease  in 

Larvae  of  bees. 

Cheshire  and 

diating      from      centre 

bees     called     "  foul 

Cheyne. 

downward;  on  potato  a 

brood." 

dry  yellow  layer. 

"  Fire-blight  "  in  pear 

Burrill. 

trees. 

Liquefy     gelatin  ;      grow 
only   in  atmosphere   of 

Causes  quarter  evil  in 
animals. 

Blood  and  tissues. 

Bellinger. 

hydrogen. 

Liquefying;  granular  col- 

Causes splenic  fever  in 

Found   in    tissues 

Rayer      and 

onies  with  irregular  bor- 
der;   on    potato    a  dry, 
creamy  layer;  in  test-tube 

animals;    malignant 
pustule  in  man. 

and    excreta    of 
diseased  animals. 

Davaine. 

a  thorny,  prickly  track. 

Grows  well  on  gelatin  ;  pale- 

Fatal  in  mice  and  rab- 

Mucous membrane 

Loffler     and 

gray  colonies  ;  not  lique- 

bits. 

of  diphtheria. 

Cohii. 

fying;   slow  growth  on 

potato. 

Causes  "  flacherie  "  in 

Intestines  of  silk- 

Bechamp. 

silkworms. 

worms. 

Gelatin  colonies  appear  as 
small     semi-transparent 

Sausage      and      meat 
poisoning. 

Intestine  of  pig. 

Van  Ermen- 
gem. 

spheres. 

Does  not  liquefy  gelatin  ; 
white,  point-like  colonies 

Causes  bubonic  plague. 

Tissues,           body 
fluids,  and  secre- 

Yersin    and 
Kitasato. 

turning  gray  and  then 

tions   of  plague 

brown. 

patients. 

Causes  dental  caries. 

Teeth  slime. 

Robin. 

Plague. 

Not  liquefying;  irregular 
scale-like  colonies,  mak- 

Kills guinea  pigs. 

Human  faeces. 

Brieger. 

ing  the  gelatin  viscid. 
Liquefying;  opaque  centre 
with  ragged  periphery  ; 

Causes  "  black  leg,"  or 
Rauschbrand,  in  cat- 

Animals    affected 
with  disease. 

Arloing,  Car- 
nevin,  and 

in  test-tube  growth  be- 

tle. 

Thomas. 

low,  with  gas  formation. 

264 


CHIEF    CHARACTERISTICS 


PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

CHOLERA  ASIATICS 

Spirillum. 

Mottfe  spiral-shaped 
rods,  often  in  chains  ; 
very  short  flagella  on 
ends,  and  strictly 
aerobic;  spores  have 
not  been  found. 

3tomaine-like  mus- 
carine  ;  and  tox- 
albumin,  soluble 
in  water. 

CHOLERA     GALLI- 
NARUM    (chicken 
cholera). 

Bacillus. 

Immotile,  cocci  -  like 
rods  ;  without  spores  ; 
strictly  aerobic. 

Toxalbumin. 

CHOLERA  NOSTRAS 
(Finckler). 

Spirillum. 

Motile,  comma-shaped 
rods  ;  strictly  aerobic. 



COLI  COMMUNIS. 
CRASSUS      SPUTIG- 

Bacillus. 
Bacillus. 

Short  motile  rods, 
slightly  curved,  with- 
out spores;  faculta- 
tively anaerobic. 

Short,  thick  rods  with 



ENUS. 

DECALVANS. 

Micrococcus. 

rounded  ends. 
Spherical  cells  in  great 

numbers. 

DENTALIS       VIRI- 

DANS. 

Bacillus. 

Slightly  curved  rods, 
round  ends. 

Gray  pigment. 

DIARRHEA  OF  IN- 
FANTS. 

Bacillus. 

Motile,  medium-sized 
rods  ;  spores  ;  aerobic. 

Toxalbumin. 

DlARRH(EA     OF 

MEAT-POISONING. 
DIPHTHERIA. 

OlPHTHERIA          OF 

Bacillus. 
Bacillus. 
Bacillus. 

Rods  in  groups  of  two 
and   singly  ;    round 
ends;  spores. 
Immotile,  middle-sized 
rods,  rounded  ends; 
facultat.  anaerobic. 
Long  rods  in  threads. 

Toxalbumin. 

CALVES       (Vitu- 
lorum). 
DIPHTHERIA        IN 

Bacillus. 

Short  rods  in  groups. 

PIGEONS  (Colum- 
barum). 

Ronilluc 

Non-motile  *      usually 

DlPLO  BACILLUS  OF 

CONJUNCTIVITIS. 

r>aciiius. 

occurs  in  pairs. 

OF   THE    PRINCIPAL    BACTERIA. 
BACTERIA.— CONTINUED. 


265 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Liquefying    slowly,   small 
depressed    scars    giving 

Causes  cholera  Asiatica 
in  man  and  a  similar 

Faeces   of   cholera 
patients. 

Koch. 

a  frosted  appearance,  or 

trouble  in  animals. 

like  ground  glass  ;  on  po- 

tato, a  thin  brown  layer; 

in    test-tube,  a    funnel- 

shaped  liquefaction,  with 

a  bubble  of  air  in  the  top, 
the  funnel  taking  six  or 

seven  days  to  form  well. 

Not  liquefying;  small  iso- 

Causes chicken  cholera 

Blood  and  faeces  of 

Pasteur. 

lated  white  disks  ;  in  test- 

in  fowls;  not  acting 

diseased  fowls. 

tube,  a  granular  track; 

on  man. 

very  faint. 

Liquefying    rapidly;    col- 

Harmless in  man  ;  fatal 

Faeces   of   cholera 

Finckler  and 

onies  yellow-brown  thick 

to  guinea  pigs. 

nostras  and  caries 

Prior. 

masses  ;  in  test-tube,  fun- 

of teeth. 

nel  formed  in  24  hours, 

dissolving  all  gelatin  in 

two  days;  profuse  gray 

mass  on  potato. 

Not  liquefying;  dark  cen- 
tre, undulated  periphery; 

Fatal   to   guinea   pigs 
and  rabbits;   causes 

Faeces   of  nursing 
infants;    water; 

Escherich. 

green-colored    layer    on 

diarrhoea    in    man  ; 

choleraic  stools. 

potato;   milky  layer  on 

ferments  sugar. 

surface  of  test-tube. 

Not  liquefying;  oval  gray- 
ish, slimy  colonies;  nail- 

Mice  and  rabbits  die  in 
48  hours  with  gastro- 

Sputum. 

Kreibohm. 

shaped  growth  in    test- 

enteritis. 

tube. 

Causes  alopecia  areata. 

In  roots  of  hair. 

Thin. 

Not     liquefying;     round, 
sharply  -  outlined      col- 

Septic   processes    and 
death  in  mice   and 

In  caries  of  teeth. 

Miller. 

onies,  with   bluish-gray 

pigs. 

opalescence. 

Not  liquefying  ;  green  col- 
onies with  foul  odor. 

Causes  green  diarrhoea 
in  animals  when  in- 

Faeces  of    infants 
suffering      from 

Lesage. 

travenously  injected, 

green  diarrhoea. 

and  is  the  cause  of 

green    diarrhoea   in 
infants. 

Causes   death  in  ani- 

Blood and  juices  of 

Klein. 

mals,  with  symptoms 
of  septicaemia. 

choleraic     diar- 
rhoea. 

Not  liquefying;  little  yel- 
lowish colonies  ;  a  mem- 

Gives rise  to  diphtheria 
in  man  and  animals. 

Diphtheritic   exu- 
date. 

Loffler. 

branous  layer  on  potato. 

When    inoculated    in 

Diphtheritic  mem- 

Loffler. 

mice  causes  death. 

brane  of  calf. 

Whitish  patches. 

Necrosis  in  pigeons  and 

Diphtheritic  mem- 

Loffler. 

other  animals. 

brane  in  pigeons. 

Addition    of    blood-serum 

Found     in     subacute 

Conjunctival  secre- 

Morax. 

to      media      necessary  ; 

conjunctivitis. 

tion. 

liquefying. 

266 


CHIEF   CHARACTERISTICS 


PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

PUCK  CHOLERA. 

Bacillus. 

Similar  to  chicken  chol- 

DYSENTERIAE. 

DYSENTERY      (epi- 
demic). 

Bacillus. 
Bacillus. 

Bacillus 

era  bacillus;  inimo- 
tile. 

Resembles        typhoid 
bacillus. 
Short  motile  rods  ;  very 
thin. 

First  slightly  acid, 
then  alkaline. 

ERYSIPELAS        OF 
SWINE  (Rothlauf; 
rouget  du  pore). 

F<ETIDUS  OZJENX. 
FROG  PLAGUE 

Bacillus. 
Bacillus. 
Bacillus 

bacillus. 

Small,  slender   motile 
rods  ;     facultatively 
anaerobic. 

Short   rods,  very  mo- 
tile;    in    pairs   and 
chains. 

See  Swine  Plague. 

Two  vaccines,  which 
give  immunity. 

Foul  gas. 

Bacillus 

short  rods    in    one; 
thread  also  spiral. 
Short  thick  rods  with 

ENES. 

Bacillus 

rounded  ends. 
Slender  immotile  rods, 

Mallei). 

GONORRHCEJE(GOIM)- 

usually  singly;  spores; 
facultatively    anaero- 
bic. 
Diplococci         kidney- 

coccus). 
GROUSE  DISEASE 

Bacillus 

shaped  ;    motile  ;  do 
not  color  with  Gram. 

H.3SMATOCOCCUS 

Diplococcus 

cocci  in  chains;  ini- 
motile. 

Cocci  seldom  in  chains  • 

BOVIS. 

Micrococcus 

surrounded  by  a  pale 
zone. 

NATORUM. 

OF   THE    PRINCIPAL    BACTERIA. 
BACTERIA.— CONTINUED. 


267 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Small    round   yellow   col- 

Fatal for  ducks,  but  not 

Blood  of  diseased 

Cornil      and 

onies  like  wax-drops  ;  not 
liquefying. 

for  chickens  or  pig- 
eons ;  less  active  than 

ducks. 

Toupet. 

chicken         cholera; 

causes  diarrhoea  and 

exhaustion. 

Resembles  typhoid    bacil- 

Produces one  variety 

In          dysenteric 

Shiga. 

lus  in  many  respects. 

of  dysentery. 

stools. 

Not  liquefying  ;  concentri- 

The cause  of  epidemic 

In  faeces  and  mes- 

Chantemesse 

cally-arranged  colon  es  ; 

dysentery  in    man  ; 

enteric  glands. 

and  Widal. 

dry  yellow  membrane  on 

enteritis  in  guinea- 

potato. 

pigs. 

Resembles  typhoid  except 

Produces  enteritis    in 

Intestinal        con- 

Gaertner. 

that    it    ferments    dex- 

man and  animals. 

tents;   its  toxin 

trose. 

in   meat  of  dis- 

eased animals. 

Very  delicate   silver-gray 
clouds  on  the  gelatin,  like 

Causes    erysipelas    in 
swine  and  other  ani- 

Blood and  organs 
of  diseased  ani- 

Loffler. 

bone-cells  ;    not  liquefy- 

mals ;    the    German 

mals. 

ing;  in  test-tube  a  very 

"Rothlauiy  French 

faint  clouding. 

"  rouget  du  pore." 

Small     greenish     colonies 

Mice  are  killed  by  in- 

Secretion   of   per- 

Hajek. 

which  soon  become  lique- 

jection ;    rabbits    af- 

sons      suffering 

fied    and    indistinguish- 

fected with  progres- 

from ozsena. 

able  ;   a    foul    odor  pro- 

sive gangrene. 

duced. 

Eberth. 

Grayish  colonies  with  foul 

Gangrenous  tissue. 

odor. 

Causes  caries  of  teeth. 

Diseased   teeth  of 

Miller. 

animals. 

Growth  rapid  ;  liquefying  ; 
round  colonies,  visible  to 

Fatal  to  mice,  with  sep- 
tic processes. 

Suppurating   pulp 
of  tooth. 

Miller. 

naked  eye  in  24  hours. 

Light  yellow,  like  honey, 
colonies,    turning     red- 
brown  in  a  few  days. 

Glanders  is  caused  by 
the  bacillus  in  man 
and  animals. 

In  epithelium  and 
ulcerated  glands. 

Loffler. 

Grow  on  blood-serum. 

Gonorrhoea  in  man. 

Gonorrhceal    pus; 

Neisser. 

in  pus-cells  and 

epithelium. 

Not  liquefying  ;  small  scales 

Fatal    for    mice    and 

In  blood  and  or- 

Klein. 

which  turn  gray  in  a  few 
days,  the  edges  serrated. 

guinea  pigs. 

gans  of  diseased 
grouse. 

Best  at  38°  C.  ;  not  liquefy- 

Fatal for  rabbits  and 

Blood   and   organs 

Babes. 

ing;  small  white  points  ; 
sparse  grow  th  on  potato  ; 
transparent. 

rats;   hypereemia  of 
lungs    and     spleen; 
blood  -  exudate     in 

of    animals    dis- 
eased with  haemo- 
globinuria. 

peritoneal  cavity. 

Supposed    to    be    the 

Found  in  this  dis- 

Klebs. 

cause  of  the  disease. 

ease. 

268 


CHIEF    CHARACTERISTICS 


PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

H^EMORRHAGIC 

SEPTICAEMIA  (In- 
fectious   PI  euro- 
pneumonia,  Wild 
Plague,    German 
Swine       Plague, 
Cattle       Plague, 
Steer  Plague.Rab- 
bit  Septicaemia). 
HOG  CHOLERA 
(Swedish     swine 
plague). 

INFLUENZA 

Bacillus. 

Bacillus. 
Bacillus. 

Short   rods,   twice   as 
long  as  broad;  im- 
motile. 

Very  motile  oval  rods, 
similar    to    hsemor- 
rhagic  septicaemia. 

Very  minute  rods  or 

Peptonizes     milk 
without  coagula- 
tion. 

INSECTORUM. 

INTRACELLULARIS 
MENINGITIDIS. 

KOCH-WEEKS 

Micrococcus. 
Diplococcus. 

in  clumps. 

Oval  cells  in  chains  and 
zoogloea  ;  streptococci. 
Resembles  gonococcus 
in   morphology  and 
arrangement  in  inte- 
rior of  leucocytes. 



LACTIS  ./EROGENES. 

Bacillus. 

bacillus. 
Short,  thick  immotile 

LEPRJE 

Bacillus 

rods. 

LlQUEFACIFNS  CON- 

Micrococcus 

with  pointed  ends. 
Single  cocci  •  never  in 

JUNCTIVE. 

LUPUS. 

MALIGN  ANTCE  DEM  A 
(Gangrenous  Sep- 
ticaemia,    Vibrio 
Septique). 

MAMMITIS  OF  Cows. 

Bacillus. 
Bacillus. 

Micrococcus 

threads. 

Same  as  Tuberculosis. 

Large,    slender    rods, 
rounded  ends,  often 
in   threads  ;    motile, 
with     flagella     and 
spores  ;          strongly 
anaerobic. 
Oval  cocci  in  chains; 

Soluble  vaccine. 

streptococci  ;     facul- 
tatively anaerobic. 

OF    THE    PRINCIPAL    BACTERIA. 
BACTERIA.— CONTINUED. 


269 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

White     isolated     pinhead 

A  disease  having  dif- 

Blood and  serum  of 

Hueppe. 

points,  not  growing  on 

ferent  names  in  dif- 

diseased animals. 

potato;    best  at  37°  C.; 

ferent  animals,  cha- 

not liquefying. 

racterized  by  oedema, 

haemorrhage,       and 

septicaemia. 

Very  good  growth  on  gel- 
atin and  potatoes  ;  a  yel- 
low-brown color. 

In    experiment,    ani- 
mal's death  in  four 
to  eight  days;   bac- 

Not spread  through 
tissue,  but  in  ca- 
pillaries of  dis- 

Salmon   and 
Selander. 

teria  in  little  emboli 

eased  swine. 

in  capillaries. 

Gi'ow  best  on  blood-agar; 
colonies      very      small, 

Produces  epidemic  in- 
fluenza. 

Secretions  of  respi- 
ratory tract. 

Pfeiffer, 
Kitasato, 

almost  transparent. 

Canon. 

A   contagious   disease 

Stomach  of  chinch- 

Burrill. 

in  the  chinch-bug. 

bug. 

Transparent          colonies, 

Causes  epidemic  cere- 

In     cerebro-spinal 

Weichsel- 

forming  thin    layer  on 

bro-spiual      menin- 

fluid  and    nasal 

baum. 

LotHer's        blood-serum 

gitis. 

secretions. 

and  glycerine  agar. 

Rarely  grows,  except   on 

Most  common  cause  of 

Conjunctival      se- 

Koch      and 

serum  agar. 

acute         contagious 

cretion. 

Weeks. 

conjunctivitis. 

Small  porcelain-lite  disks 
with   depressed   centre; 

Fatal  to   guinea   pigs 
and  rabbits  ;  coagu- 

Faeces of  nursing 
infants    and    of 

Escberich. 

funnel-shaped     in    test- 

lates   milk;   decom- 

cholerine. 

tube  with  gas. 

poses    sugary    solu- 

tions. 

On    blood  -  serum    round 
white    plaques  with  ir- 

Causes leprosy  in  man 
and  animals. 

Leprous  tissue. 

Hansen. 

regular  borders. 

Liquefying;  growth  rapid; 

On   cornea  of  rabbits 

Normal        human 

Gombert. 

colonies  on  surface,  with 

causes  slight  cloud- 

conjunctiva. 

little  radiating  branches 
from  a  dark  centre  ;  those 

ing. 

in  deep,  berry-shaped. 

Liquefying;  thick  centre, 

Animals    quickly   die 

Garden-earth. 

Pasteur. 

radiating  periphery  ;  in 

with  extensive  gan- 

high culture  in  test-tube, 

grene  and  oedema. 

gas-bubbles   arise,    with 

foul  odor. 

Not    liquefying;     brown, 
round  granular  colonies; 

Causes  contagious  rnain- 
mitis  in  cows  ;  coagu- 

Mammary gland. 

Nocard     and 
Mollereau. 

grows  slowly;  in  test-tube, 

lates  milk. 

heavv  deposit  along  ""he 

needle's  track. 

270 


CHIEF    CHARACTERISTICS 


PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

MAMMITIS    OF 
SHEEP. 

MELITENSIS. 

Micrococcus. 
Micrococcus. 

Streptococci     and     in 
fours. 

5  jix  in  diameter  •  occurs 

METCHNIKOVI. 
NEAPOLITANUS. 
NOM.E. 

OXYTOCUS     PERNI- 

Spirillum 
(vibrio). 

Bacillus. 
Bacillus. 

Bacillus 

singly  or  in  chains 
of  two  or  more  ;  said 
to  be  flagellated. 

Motile     spirals     with 
flagella;  aerobic. 

Small   iromotile   rods, 
with  rounded  ends: 
no   spores  ;    faculta- 
tively anaerobic. 
Small  rods,  with  round- 
ed ends,  growing  of- 
ten in  long  threads. 

Short  rods  with  round 

An  alkaline  vac- 
cine which  will 
cause  immunity. 

Produces  acids  in 
gelatin  cultures. 

CIOSUS. 

PARATYPHOID. 
PNEUMONIA  (Pneu- 

Bacillus. 
Bacillus 

ends. 

Resembles        typhoid 
bacillus. 

Short    im  mo  tile  rods 

Indol  sometimes 
produced. 

mococcus  of  Fried- 
lander). 

PNEUMONIA  (Pneu- 

Bacillus. 

singly  or  in  diplococ- 
ci,  surrounded  with 
capsule  ;    no  spores  ; 
not     colored     with 
Gram;  facultatively 
anaerobic. 
Short,  oval  rods,  often 

mococcus  of  Fran- 
kel  ;    Micrococcus 
of  Pasteur). 

PNKUMONICIS 

Bacillus 

in  chains  ;  immotile  ; 
no  spores  ;  in  the  tis- 
sue surrounded  with 
capsule,  colored  with 
Gram  :  facultatively 
anaerobic. 
Short  thick  motile  rods 

-- 

AGILIS. 

PROTEUS  SEPTICUS. 

Bacillus. 

in  pairs. 

Slightly  curved    rods, 
swelled  in  portions, 
sometimes    in    long 
threads;  motile. 

Foul  gas. 

OF    THE    PRINCIPAL    BACTERIA. 
BACTERIA.— CONTINUED. 


271 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Liquefying;  round  centres 

Causes  contagious  gan- 

Found in  the  milk 

Nocard. 

with  zone    of   liquefac- 

grenous     mammitis 

of          diseased 

tion  ;      cone-shaped     in 

in  sheep. 

sheep. 

test-tube. 

Small,      round,      slightly 

Causes  Malta  fever. 

Best  obtained  from 

Bruce. 

raised    disks;     do     not 

spleen. 

liquefy. 

Grows    quickly;   colonies, 
some  like  cholera  Asiat- 
ica,  others  like  cholera 

Causes   vibrion    septi- 
caemia in  guinea  pigs 
and  pigeons. 

Faeces  of  fowls. 

Gamaleia. 

nostras;  liquefying. 

Not  liquefying  ;  thin  pearl- 
like  scales  in  several  lay- 

Causes death  in  some 
animals  :     not     the 

Cholera    epidemic 
of  Naples,  1884. 

Emmerich. 

ers;  wrinkled  and  mu- 

cause of  cholera. 

cous  layers  on  potato. 

Granular  spherical  colonies 

No  action  on  mice  or 

In  necrotic  tissue 

Schimmel- 

in  the  deep,  flat  on  the 

rabbits. 

of  noma. 

busch. 

surface;  not  liquefying; 

growth  rapid;  best  at  35° 
C. 

Small  yellow  granular  col- 
onies;    nail-culture    in 

Intravenous  injection 
causes  death  in  mice 

Sour  milk. 

Wyssokow 
itsch. 

test-tube. 

and   rabbits;    turns 

milk  acid. 

Ferments  glucose,  but  not 

Causes           continued 

Intestinal       con- 

Widal.Gwyn, 

lactose     or    saccharose  ; 

fevers. 

tents. 

Schott- 

does  not  coagulate  milk. 

miiller. 

Does   not  liquefy;   grows 
quickly;    a    button-like 

An  accompaniment  of 
pneumonia,     not     a 

Pneumonic       and 
other      sputum, 

Friedlander. 

colony;  in   test-tube,  as 

cause  ;   animals   not 

and  lung  tissue. 

if  a  nail  driven  in  the 

affected. 

gelatin  with  head  on  sur- 

face. 

Does    not    liquefy;    grows 
slowly  ;    small,    well-de- 

Causes pneumonia  in 
man,  septicaemia   in 

Sputum  of  lung  af- 
fections and  se- 

A. Frankel. 

fined  masses;in  test-tube, 

animals;  also  serous 

rous    inflamma- 

little separate  globules, 

inflammations       in 

tions. 

one  above  the  other. 

man,  as  pleurisy,  per- 

itonitis, etc. 

Liquefying  ;  dark  granular 
colonies  ;  thick  sediment 

Pneumonia  in  rabbits. 

From  rabbits'  pneu- 
monia. 

Schon. 

in  test-tube. 

Growth  rapid  ;  liquefying  ; 
colonies  have  foul  odor, 
are  small,  thick  branches, 
but  soon  all  liquid. 

Fatal  for  mice  in  one 
to  three  days. 

From  a  child  dying 
of  intestinal  gan- 
grene. 

Babes. 

272 


CHIE2    CHARACTERISTICS 


PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

PSITTACI       (perni- 
ciosus). 
PYOCYANEUS. 

PYOCYANEUS  /3. 

Micrococcus. 
Bacillus. 

Bacillus. 

Streptococci  and  zoog- 
loea. 
Thin  motile  rods;  fa- 
cultatively anaerobic. 

Forms  a  brown-yellow 

Pyocyanin,  a  non- 
poisonous  pig- 
ment. 

PYOGENES  (Strepto- 

Micrococcus. 

pigment  ;    otherwise 
identical  with  above. 
Streptococci  and  zoog- 

coccus  erysipela- 
tis—  Fehleisen). 
PYOGENES  ALBUS. 

Micrococcus. 

loea. 
Staphylococci          and 

PYOGENES  AUKEUS 
(micrococcus     of 
osteomyelitis- 
Becker). 

PYOGENES  CITREUS. 

Micrococcus. 
Micrococcus. 

streptococci  ;     facul- 
tatively anaerobic. 
Staphylococci  and  zoog- 
loea  ;       facultatively 
anaerobic. 

Same  as  Pyogenes  au- 

Ptomaine,  toxal- 
bumin,  and  pig- 
ment. 

PYOGENES  FCETIDUS. 

Bacillus 

retis. 

PYOGENES  TENUIS. 

Micrococcus. 

pairs. 
Cocci  without  definite 

Spirillum 

arrangement. 

(Obermeier). 

motile. 

OF    THE    PRINCIPAL    BACTERIA. 
BACTEKIA.— CONTINUED. 


273 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Causes  disease  in  gray 

In    blood   of  par- 

Worff. 

parrots. 

rot's  disease. 

Liquefying;  large,  flat  col- 
onies with  greenish  fluo- 

Fatal for  animals;  col- 
ors    the     dressings 

Pus. 

Gessard. 

rescence  ;  on  potato,  yel- 

green. 

low-green    skin,    deeply 
coloring  the  pulp. 

Ernst. 

Not      liquefying;      round 
punctiform  colonies;  slow- 

Suppuration   and  sep- 
ticaemia in  animals. 

Pus. 

Rosenbach. 

growing. 

Liquefying;  white  opaque 

Suppuration    and   ab- 

Pus. 

Rosenbach. 

colonies. 

scess. 

Liquefying;  small  colonies 
with  a  yellow-orange  pig- 

Causes  abscesses   and 
suppuration  in  man 

Pus. 

Rosenbach. 

ment  in  centre;    yeast- 

and  animals. 

like  smell  ;  a  moist  layer 

on  potato. 

Colonies,         citron  yellow 

Suppuration. 

Pus. 

Passet. 

color. 

Not    liquefying;     mucous 

Fatal  to  animals. 

Pus. 

Passet. 

layer    on    potato  ;    very 

thick;    in    test-tube,    a 

slight  layer  on   surface, 

and  small  points  along 

the  track. 

On    surface,   transparent; 

Pus  of  abscesses. 

Rosenbach. 

thin      growth;       grows 

slowly. 

Cannot  be  cultivated. 

Causes  fever   in    man 

Blood  of  man  dur- 

Obermeier, 

and  animals,  and  is 

ing  an  attack  of 

the  cause  of  relapsing 

the  disease. 

fever. 

274 


CHIEF    CHARACTERISTICS 


PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

RHINOSCLEKOMA. 

SALIVARIUS    PYOG- 
ENES. 


SALIVARIUS  SEPTI- 
CUS. 


SALIVARIUS   SEPTI- 

cus. 


SAPROGENES  No.  II 


SAPROGENES  No.  Ill, 


SAPROGENES  FCETI- 
DUS. 


SENILE  GANGRENE. 


SEPTICAEMIA  AFTER 
ANTHRAX. 

SEPTICAEMIA        OF 
MICE. 


SEPTICAEMIA  OF 
RABBITS  (Cuni- 
culicida). 

SEPTICUS  ACUMINA- 

TUS. 


Bacillus. 
Micrococcus. 


Bacillus. 


Micrococcus. 


Bacillus. 


Bacillus. 


Bacillus. 


Bacillus. 


Micrococcus. 


Bacillus. 


Bacillus. 
Bacillus. 


SEPTICUS 

EN  US. 


AGRIG-  I  Bacillus. 


See    Pneumococcus    of 

Very  small  round  cocci 
and  staphylococci. 

Short,  immotile  rods, 
encapsulated  in  pairs, 
sometimes  long 

chain ;  aerobic. 

Cocci  singly  and  in 
zoogloea;  aerobic. 

Short  rods ;  faculta- 
tively anaerobic. 


Very  short  rods :  facul- 
tatively anaerobic. 

Immotile  rods ;  spores. 


Thin  rods;  immotile; 
singly  and  in  pairs; 
ends  somewhat  thick- 
ened ;  aerobic ;  spores. 

Motile  streptococci. 


Smallest  bacillus 

known ;  immotile. 


See  Hcemorrhagic  Septi 


Thin,      lancet-shaped 
rods ;  very  slender. 


Very  short  rods. 


Friedlander,   with 


Foul  gas. 


Foul  gas. 


Foul  gas. 


ccemia. 


OF    THE    PRINCIPAL   BACTERIA. 
BACTERIA.— CONTINUED. 


275 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

which  it  is  identical. 



, 

Frisch. 

Slowly  liquefying;  small 
white  opalescent  col- 
onies. 

Local  abscess  in  ani- 
mals. 

Saliva. 

Biondi. 

Not  liquefying;  gray  cir- 
cular colonies;  trans- 
parent zone  ;  in  test-tube, 
separated. 

Fatal  to  animals. 

Saliva  of  healthy 
persons. 

Biondi. 

Not  liquefying;  round  col- 
onies ;  separated  dots  in 
test-tube. 

Fatal  to  animals. 

Saliva  of  puerperal 
women. 

Biondi. 

Grows  quickly  ;  on  agar, 
hyaline  drops  which 
quickly  coalesce,  and 
form  a  mucoid  layer  with 
a  foul  odor,  that  of  per- 
spiring feet. 

Produces  septicaemia  in 
rabbits. 

Sweat  of  feet. 

Rosenbach. 

Forms  a  fluid  gray  band  on 
agar;  odor  of  putrefac- 
tion. 

Suppuration  in  rabbit. 

Putrid  marrow  of 
bone. 

Rosenbach. 

Not  liquefying;  thin,  trans- 
parent layer  ;  putrid  odor. 

Rabbits  killed  with 
large  doses. 

Mesenteric  glands 
of  swine  with 
erysipelas  and  of 
healthy  swine. 

Schottelius. 

Round  yellow  colonies; 
liquefying  in  36  hours; 
best  growth  at  37°  C. 

Causes  gangrene  in 
mice,  similar  to  se- 
nile gangrene  of 
man. 

In  gangrenous  tis- 
sue and  blood  of 
senile  gangrene. 

Tricomi. 

In  bouillon  virulence  de- 
stroyed. 

Septicaemia  in  rabbits, 
but  not  in  chickens 
or  guinea  pigs. 

Blood  of  animal 
dead  from  an- 
thrax. 

Charrin. 

Not  liquefying  ;  small  floc- 
culent  masses  in  the  deep; 
grows  very  slowly;  in  the 
test-tube  producing  a 
faint  cloud. 

Septicaemia  in  house- 
mice,  but  not  field- 
mice. 

Putrefying  liquids. 

Koch. 

At  37°  C.  on  blood-serum 
small  transparent  plates  ; 
later  on,  turning  yellow. 

Pathogenic  for  rabbits 
and  guinea  pigs;  fever; 
and  bacilli  in  blood 
and  organs. 

Navel  stump  of 
child  dead  of 
septicaemia. 

Babes. 

Not  liquefying  ;  brown  cen- 
tre, a  ring,  then  yellow 
zone. 

Septicaemia  in  mice  and 
rabbits. 

Earth  of  recently- 
ploughed  fields. 

Nicolaier. 

2/6 


CHIEF    CHARACTERISTICS 


PATHOGENIC 


Name. 

Genus. 

Biology. 

Product. 

SEPTICUS  LIQUEFA- 

CIENS. 

Micrococcus. 

Streptococci  and  diplo- 
cocci. 



SEPTICUS  ULCERIS. 

Bacillus. 

Oval  rods  ;  motile. 

Gas  ;  no  odor. 

SEPTICUS  VESICLE. 

Bacillus. 
Bacillus 

Rods    always    single; 
very   motile;      oval 
spores. 

Slender     curved  rods, 



SOFT  CHANCRE. 

Bacillus. 

identical  with  what 
was  known  as  syph- 
ilis bacillus  of  Lust- 
garten. 

Minute      oval      rods, 

SPUTIGENUM. 
SUBFLAVUS. 

Spirillum. 
Micrococcus. 

chiefly  in  groups  or 
chains. 
Curved,  comma  shaped 
rods;  motile. 

Dipl'icocci   like  gono- 



cocci  ;     colored     by 
Gram. 

SWINE  PLAGUE 
(American      and 
French). 

Bacillus. 

Motile,  oval  rods,  sim- 
ilar  to  that   of  hog 
cholera. 

Causes  casein  pre- 
cipitate in  milk 
and  acid  forma- 
tion. 

SYCOSIFERUS  FCETI- 

DUS. 

Bacillus. 

Short,  straight  immo- 
tile   rods,   often    in 
threads. 

On  potatoes  a  foul 
odor. 

OF    THE    PRINCIPAL    BACTERIA. 
BACTERIA.— CONTINUED. 


277 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Liquefying  ;  a  thin  gran- 
ular streak,  the  surface 
sunken  in  ;  later,  cone- 
like,  the  walls  covered 
with  leaf  -shaped  col- 
onies. 

Pathogenic    for    mice 
and   rabbits,  produ- 
cing o?dema,  in  the 
serum  of  which  the 
cocci  abound. 

Blood  and  organs 
of  child  dying  of 
septica'mia. 

Babes. 

Liquefying;  yellow  col- 
onies, taken  up  with  gas 
later  on. 

An  ulcer  in  inoculated 
animals,  followed  by 
paralysis  and  death. 

In  blood  of  child 
with  gangrenous 
ulcer. 

Babes. 

Not  liquefying  ;  small  pin- 
head  colonies,  growing 
slowly:  never  larger;  a 
brown  centre,  yellow 
periphery. 

Pathogenic    for    mice 
and    rabbits,  produ- 
cing death. 

In  urine  of  cys- 
titis. 

Clado. 

Not  cultivated. 



Normal  preputial 
secretions. 

Alvarez    and 
Tavel. 

Has  not  been  cultivated. 

Produces  soft  chancre. 

In  the  sore. 

Ducrey. 

Not  cultivated. 

Causes  death   in    ani-    In  caries  of  teeth 
mals.                                   and  saliva. 

Lewis. 

Growth  slow;  liquefying; 
on  tenth  day  yellow 
points  with  thready 
boundary;  on  potato,  a 
brown,  thread  -  like 
growth  after  two  weeks. 

No  result  on  mucous 
m<inbrane;  injected 
under   skin,  abscess 
results. 

Normal  secretion 
of  vagina  and 
urethra. 

Bumm. 

Not  liquefying;  growth 
similar  to  typhoid  germ  ; 
on  potatoes  good  growth. 

Found  in  American  and 
French  swine  plague, 
in    frog  plague,  and 
Texas  fever;"  animals 
affected  locally. 

Found  in  capil- 
laries in  little  em- 
boli;  not  spread 
in  organs  of  dis- 
eased animals. 

Billings, 
Rietsch,  and 
Eberth. 

Slow  growth  ;  not  liquefy- 
ing; after  four  days,  lit- 
tle white  points,  which 
do  not  change  for  several 
weeks,  then  the  super- 
ficial ones  are  mucous- 
like;  nail  growth;  on 
potatoes,  rapid  growth. 

On  human  skin  causes 
eruption,      vesicular 
around    hairs,    then 
it  becomes  pustular; 
similar  to  sycosis. 

From  svcosis  of  the 
beard. 

Tommasoli. 

278 


CHIEF    CHARACTERISTICS 


PATHOGENIC 


Name. 


Genus. 


Biology. 


Product. 


SYPHILIS      (Spiro- 
cheta  Pallida). 


TETANUS. 


TETRAGENUS. 


TIMOTHY  GRASS. 


TOXICATUS. 
TUBERCULOSIS. 


TYPHOID. 


TYPHOID  OF  SWINE 
(swine  plague). 

TYROGENUM 
(Deneke's). 


XEROSIS. 


Spirocheta. 


Bacillus. 


Micrococcus. 


Bacillus. 


Micrococcus. 
Bacillus. 


Bacillus. 


Spirillum 
(vibrio). 


Bacillus. 


Small  delicate  spirals, 
difficult  to  stain. 

Large,  slender  motile 
rods,  with  spores  in 
one  end,  drumstick 
shape,  often  in 
threads ;  true  ana3ro- 
bic. 

Large  round  cells,  uni- 
ted in  groups,  usual- 
ly of  four,  and  sur- 
rounded by  a  cap- 
sule ;  immotile ;  aero- 
bic. 

Extremely  acid-fast; 
resembles  tubercle 
bacillus;  in  cultures 
may  show  club  for- 
mation and  branches. 

Cocci  singly  and  in 
pairs. 

Slender  rods,  usually 
in  pairs;  not  motile; 
spores  not  definitely 
determined ;  facul- 
tatively anaerobic. 


Slender  motile  rods, 
sometimes  in  tli reads; 
flagella,  but  no  spores; 
facultatively  anaero- 
bic. 

See  Swine  Plague. 

Spiral  -  shaped  rods ; 
serobic. 


Similar  to  diphtheria 
bacillus. 


Ptomaines,  teta- 
nine,  tetanotox- 
ine,  spasmotox- 
iue;  also  a  tox- 
albumin. 


Kochine  or  parato- 
line,  a  glycerin 
extract  of  the 
pure  culture  (tu- 
berculin). 


Typhotoxin      and 
toxalbumin. 


OF    THE    PRINCIPAL   BACTERIA. 


BACTERIA.— CONTINUED. 


Culture  Characters. 

Actions. 

Habitat. 

Discoverer. 

Not  cultivated. 

Supposed  to  cause  syph- 

In tissue  and  secre- 

Schaudinn. 

ilis. 

tions    of    syphi- 

litics. 

Liquefy     gelatin     slowly  ; 
colonies    have    radiated 

Produces    tetanus    in 
man  and  auimals. 

Earth  and  manure. 

Nicolaierand 
Kitasato. 

appearance  ;     a    thorny 

. 

growth  along  the  track 

in  test-tube. 

Not  liquefying;  little  por- 
celain-like disks;   thick 

Fatal  to    guinea    pigs 
and  white  mice. 

Found  in  cavern- 
ous      phthisical 

Gaflfky. 

slimy  layer  on  potato. 

lungs. 

Colonies  visible  in  thirty- 

May    produce    tuber- 

Infusions            of 

Moeller. 

six  hours,  scale-like  and 

cles. 

timothy  grass. 

grayish  white. 

Supposed    to    be    the 

Found  in  the  Rhus 

Burrill. 

cause  of  Rhus(  poison 

toxlcodendron. 

ivy)  poisoning. 

Grows  best  on  blood-serum 

Causes  tuberculosis,  lo- 

In all  organs  and 

Koch. 

and  glycerin  agar  at  37° 
C.,  forming  little  white 
crumbs  on  the  surface; 

cal  and  general,  in 
man  and  lower  ani- 
mals. 

secretions  of  tu- 
bercular persons. 

under  microscope  a  hairy 

matted     coil     is     seen  ; 

growths      on      potatoes 

when  air-tight  have  been 

obtained. 

Not  liquefying  :  little  whet- 

Gives  rise   to   enteric 

Found    in  dejecta 

Eberth. 

stone-shaped  yellow  col- 
onies in   the  deep,  and 

or  typhoid  fever  in 
man. 

and   spleen    and 
urine  of  typhoid 

leaf-shaped  ones  on  the 

patients. 

surface  ;  on  potato,  a  very 

transparent,  moist  layer. 

Liquefy     rapidly;     small 
round     colonies;     dark 

Several   animals  have 
died    from    inocula- 

From old  cheese. 

Deneke. 

funnel-shaped    liquefac- 

tions. 

tion  in  test-tube. 

Differs     from    diphtheria 

Found    in    patho- 

Kuschbert 

bacillus  in  not  produc- 

logical       condi- 

and 

ing  acid  in  bouillon. 

tions  of  conjunc- 

Neisser. 

tiva,    sometimes 

in  normal  eye. 

INDEX 


ABBE'S  condenser,  26 
Achorion  Schb'nleinii,  210 
Acid  dyes,  30 
Aerobins,  facultative,  23 

obligate,  23 
Aerobioscope,  219 
Aeroscope,  216 
African  tick  fever,  194 
Agar-agar,  55 

blood-,  60 

bouillon,  preparation,  55 

gelatin-,  57 

glycerin-,  57 

inoculation  of,  62 

Japanese  method  of  preparing,  56 

litmus,  60 

nutrient,  preparation  of,  55 
Age,  influence  of,  on  bacteria,  25 
Agglutination  phenomenon,  131 

reaction  for  tubercle  bacillus,  119 
Agglutinins,  81 
Agglutinogen,  82 
Aggressins,  77 
Air,  bacteria  in,  216,  219 

examination  of,  216 
Alcoholic  solution,  saturated,  32 
Alkaline  anilin-water  solutions,  34 

methylene-blue,  32 

stains,  31 

Amoeba  dysenteriae,  195 
Anaerobins,  facultative,  24 

obligate,  23 
Anilin  dyes,  30 
Anilin-oil  water,  31,  32 
Anilin-water  dyes,  32 
Animals  as  culture -media,  67 

experiments  upon,  84 
Anthrax,  105 

symptomatic,  196 


Antisepsis,  233 

Antiseptics,  233 

Antitoxin  of  diphtheria,  128 

of  pneumonia,  153 

of  tetanus,  173 
Antituberculous  serum,  119 
Arnold's  steam  sterilizer,  47 
Artesian  well  water,  221 
Arthrospores,  20 
Arthrosporous  bacteria,  22 
Asbestos,  221 
Asexual  cycle  in  man,  185 
Aspergillus  flavus,  211 

fumigatus,  211 

glaucus,  211 

Asporogenic  bacteria,  22 
Autoclave,  47 
Autovaccines,  170 


BACILLEN  emulsion,  119 
Bacillus,  17 

acidi  lactici,  95 

aerogenes  capsulatus,  180 

alvei,  204 

amylobacter,  97 

anthracis,  105 

avicidus,  198 

Boas-Oppler,  96  ' 

botulinus,  139 

butyricus,  96 

capsule,  156 

coeruleus,  99 

coli  communis,  137 

diagnostic  points  of,  225 
in   water,    examination    for, 
224 

enteritidis  sporogenes,  182 

erythrosporus,  99 

281 


282 


INDEX 


Bacillus,  feces,  135 
fluorescens,  163 

liquefaciens,  100 
geniculatus,  96 
hay,  94 
indicus,  92 
Koch- Weeks',  165 
lactis  cyanogenus,  97 

erythrogenes,  98 
lepra,  120 
mallei,  121 
megaterium,  93 
melitophtharus,  204 
mesentericus  vulgatus,  92 
Milzbrand,  105 
murisepticus,  202 
mycoides,  93 
Neapolitanus,  135 
cedematis  maligni,  174 
of  anthrax,  105 
of  bluish-green  pus,  163 
of  bubonic  plague,  177 
of  chicken  cholera,  198 
of  cholera,  140 

in  water,  143 

products  of,  142 
of  diphtheria,  123 

products  of,  127 
of  dysentery,  179 
of  erysipelas  of  swine,  201 
of  fowl  septicemia,  198 
of  glanders,  121 
of  influenza,  157 
of  leprosy,  120 
of  malignant  pustule,  105 
of  red  milk,  98 
of  rhinoscleroma,  153 
of  soft  chancre,  177 
of  splenic  fever,  105 
of  symptomatic  anthrax,  196 
of  syphilis,  120 
of  tetanus,  170 
of  typhoid  fever,  129 

bacteria  resembling,  135 
in  blood,  136 
in  water,  135 
products  of,  136 
paracolon,  136 
paratyphoid,  136 
phosphorescens  gelidus,  101 

indicus,  100 


Bacillus  phosphorescens  indigenus, 
100 

prodigiosus,  91 

psittacosis,  137 

pyocyaneus,  163 
B,  165 

ramosus,  93 

root,  93 

smegma,  120 

spinosus,  95 

subtilis,  94 

tuberculosis,  109 
products  of,  118 

violaceus,  99 

Wurzel,  93 
Bacteria,  17 

asporogenic,  22 

action  in  causing  disease,  75 

aerobic,  23 

anaerobic,  23 

cultivation  of,  71 
Botkin's  method    74 
Buchner's  method,  74 
Esmarch's  method,  72 
Frankel's  method,  73 
Hesse's  method   72 
Liborius's  method,  71 
Roux's  method,  73 
Wright's  method,  74 

arthrosporous,  22 

causing  disease,  25 

colonies  of,  growth  and  appear- 
ance, 68 

distribution  of,  22 

effects  of,  general,  76 
local,  76 

examination  of,  methods,  26 

fluorescence  of,  25 

fluorescent,  99 

gas-forming,  25 

growth  of,  23 

in  air,  216,  219 

in  blood,  232 

in  conjunctiva,  230 

in  ear,  231 

in  genito-urinary  passages,  232 

in  intestine,  231 

in  milk,  95 

in  mouth,  230 

in  nasal  cavity,  231 

in  skin,  229 


INDEX 


Bacteria  in  soil,  227 
in  stomach,  231 
in  urethra,  168 
in  urine,  102 
in  vagina,  168 
in  water,  220,  223 
infectious,  75,  76 
influence  of  age  on,  25 

of  electricity  on,  24 

of  light  on,  24 

of  oxygen  on  life  and  growth, 

23 

of  Rontgen  rays  on,  24 
of    temperature    on    life    and 

growth,  23 
life  of,  23 
locomotion,  19 
method  of  counting,  in  a  culture, 

87 
non-pathogenic,  25,  91 

in  water,  99 

table  of,  238-261 
odors  from,  25 

of  hemorrhagic  septicemia,  200 
of  milk,  228 
of  pneumonia,  148 
of  suppuration,  158 
origin,  22 
oxidation  by,  24 
parasitic,  _  75 
pathogenic,  25,  75,  105 

for  animals,  196 

table  of,  262-279 
phosphorescence  by,  25 
phosphorescent,  100 
pigmentation  by,  25 
pyogenic,  76 
reduction  by,  24 
reproduction,  19 
saccharolytic,  139 
saprophytic,  75 
sewage,  examination  for,  224 
specific,  76 
staining  of,  30 
suppurative,  76 
tables  of,  238-279 
types,  17 

unstained,  examination  of,  27 
vibratory  movements,  19 
vital  action,  24 
Bacterial  treatment  of  sewage,  226 


Bactericie  du  charbon,  105 
Bacteriologic  examination  of  organs 

and  cavities,  229 
Bacteriolysis,  81 
Bacterium  acidi  lactici,  96 

aeruginosum,  163 

Balticum,  101 

Fischeri,  101 

Pfliigeri,  101 

syncyanum,  97 

termo,  205 

ureae,  102 

Zopfii,  94 
Ball's  forceps,  37 
Basic  dyes,  30 
Beggiatoa,  101 

alba,  102 
Biedert's     method     of     collecting 

tubercle  bacilli,  115 
Bile-salt  media,  MacConkey's,  61 
Blastomycetes,  207 
Blastomycosis,  208 
Blood,  bacteria  in,  232 

coagulum,  60 

typhoid  bacillus  in,  136 
Blood-agar,  60 
Blood-capsule,  Wright's,  89 
Blood-serum,  57 

coagulation  of,  58 

in  liquid  state,  preservation,  59 

mixture,  Lb'ffler's,  60 

nutrient,  preparation  of,  57 

sterilization  of,  58 
Boas-Oppler  bacillus,  96 
Boiled  eggs,  62 
Boiling  water,  222 
Borer,  Frankel's,  227 
Botkin's  method  of  cultivation  of 

anaerobic  bacteria,  74 
Bouillon,     agar-agar,     preparation 

of,  55  . 

guinea-pig,  62 

LofHer's,  49 

sterilization  of,  50 
Bouillon-gelatin,    Koch-Loffler,    53 
Bovine  farcin  du  bceuf,  215 
Bread  mash,  53 
Brood-ovens,  57 
Broth,  glucose,  50 

glycerin,  50 
Brownian  movements,  19 


284 


INDEX 


Bubonic  plague,  177 

Buchner's  method  of  cultivating 
anaerobic  bacteria,  74 

Buerger's  method  of  staining  cap- 
sule, 43 


CALMETTE'S  ophthalmic  tuberculin 

reaction,  119 
Capsule  bacillus,  156 

stain,  43 

Buerger's  method,  43 
of  Hiss,  35 
of  Welch,  35 
Carbol-fuchsin,  31 
Carbol-thionin  stain,  34 
Cell-contents,  18 
Cell-wall,  18 
Chancre,  soft,  177 
Charbon  symptomatique,   197,   198 
Charcoal  sponge,  221 
Chemotaxis,  80 
Chicken  cholera,  198 
Cholera,  chicken,  198 

immunity  of  Pfeiffer,  143 

red,  142 

Cladothrices,  212 
Cladothrix,  101 

actinomyces,  212 

dichotoma,  101 
Clostridium  butyricum,  97 
Clouding  of  gelatin,  54 
Coagulation  of  blood-serum,  58 
Colitis  contagiosa,  138 
Colonies,  growth  and  appearances, 
68 

macroscopic  appearance,  68 

microscopic  appearance,  68 
Comma  bacillus  of  cholera,  140 
Compound  solutions,  31 
Conjunctiva,  bacteria  in,  230 
Conjuntivitis,  epidemic,  165 

diplobacillus  of,  166 
Cotton  plugs  or  corks,  48 
Cover-glass  preparations,  36 
Crenothrix,  IOT 

Kiihniana,  101 
Cultivation,  44 

artificial,  44 

of  anaerobic  bacteria,  71 
Cultures,  egg,  fresh,  61 


Cultures,  glass  slide,  62 

methods  of,  44 

nail,  150 

plate,  64 

potato-,  51 

pure,  by  boiling,  67 

rolled,  67 

smear,  63 

stab,  63 

sterilization  of,  44 

stroke,  63 

test-tube,  63 

thrust,  63 
Cutting  sections,  38 


DAUGHTER-CELL,  207 
Decolorizing  agents,  31 
Deny's  B.  F.  tuberculin,  119 
Deodorant,  233 
Dextrose,  60 
Disinfectants,  44,  233 
Diphtheria,  123 

streptococcus  in,  129 

toxins  of,  128 

Diplobacillus  of  conjunctivitis,  160 
Diplococcus  albicans  amplus,    169 
tardissimus,  169 

intracellularis  meningitidis,  153 

lanceolatus,  151 

pneumonias,  149,  151 
Drying  specimens,  36 
Dunham's  peptone  solution,  61 

rosalic  acid  solution,  60 
Dysentery,  179 


EAR,  bacteria  in,  231 

Edema,  malignant,  174 

Egg,  boiled,  62 
cultures,  fresh,  61 

Ehrlich's  side-chain  theory  of  im- 
munity, 80 

Electricity,    influence   of,    on    bac- 
teria, 24 

Eisner's  typhoid  medium,  60 

Endocarditis,  160 

Endospores,  20 

Enteric  fever,  129 

Erysipelas,  160 
of  swine,  201 


INDEX 


285 


Esmarch's  cubes,  52 

method  of  cultivating  anaerobic 
bacteria,  72 

tubes,  67 
Experiments  on  animals,  84 

FECES  bacillus,  135 
Fermentation  tube,  62 

Smith's,  62 
Ferments,  25 

coagulating,  25 

diastatic,  25 

fat-splitting,  25 

hydrolytic,  25 

inverting,  25 

proteolytic,  25 
Filter,  hot-water,  54 
Filtered  water,  221 
Filtration,  236 
Finkler-Prior  vibrio,  146 
Fishing,  69 
Flagella,  19 

stain,  with  Loffler's  mordant,  43 
Fluid  media,  49 
Fluorescence,  25 
Fluorescent  bacteria,  99 
Foods  as  source  of  infection,  228 
Forceps,  Ball's, '37 
Formaldehyd,  235 
Fowl  septicemia,  198 
Frankel's  borer,  227 

method  of  cultivating  anaerobic 

bacteria,  73 

of  staining  tubercle  bacilli,  113 
Fungi,  staining,  Unna's  method,  43 

thrush,  208 

GABBET'S  acid  blue  stain,  33 
Gametes,  186 
Gas-formation,  25 
Gelatin,  53 

clouding,  54 

inoculation  of,  62 

modification  of,  55 

potato-,  60 

sterilizing,  55 
Gelatin-agar,  57 
Gelatinous  membrane,  18 
Genito-urinary    passages,    bacteria 

in,  232 


Germicides,  234 
Germination,  22 
Giemsa  stain,  35 
Glanders,  121 
Glass  plates,  64 

slide  cultures,  62 
Glossina  morsitans,  192 

palpalis,  192 
Glucose  broth,  50 
Glycerin  agar,  57 

broth,  50 
Gonococcus,  166 

urine  media  for,  61 
Gonorrhea,  166 
Gram's  iodin  solution,  33 

method  of  double  staining,  40 

of  tissue  staining,  41 
Gruber-Widal  blood-serum  test,  131 
Guinea-pig  bouillon,  62 

HANDS,  sterilization  of,  236 
Hanging  drop,  29 
Hay  bacillus,  94 
Heat,  31,  234 

as  disinfectant,  44 

dry,  45 

moist,  45 

Hemolytic  serum,  81 
Hemorrhagic  septicemia,  200 
Hesse's  method  of  collecting  bac- 
teria from  air,  216 
of   cultivating   anaerobic   bac- 
teria, 72 
Hiss'  capsule  stain,  35 

typhoid  medium,  61 
Hoffman's  pseudobacillus,  126 
Hot-air  oven,  45 
Hot-water  filter,  54 
Hueppe's  fresh  egg  cultures,  61 

IMMUNE  body,  81 
Immunity,  78 
acquired,  78 
active,  78 
Ehrlich's   side-chain    theory  of, 

80 
from     injections     of     sterilized 

products  of  bacteria,  79 
from  inoculations  of  small  doses 
of  very  virulent  bacteria,  79 


286 


INDEX 


Immunity  from  inoculations  with    | 
attenuated  or  weakened    cul- 
tures of  bacteria,  78 
inherited,  79 
natural,  78 
passive,  79 
phagocytic  or  cellular  theory  of, 

80 

theories  of,  79 
summary,  83 
unit,  129 

Impression  of  colonies,  69 
Incubators,  57 
Infection,  75 

conditions  necessary  to  produce, 

75 
Infectious  bacteria,  75,  76 

diseases,  25 
Influenza,  157 
Infusoria,  184 
Inoculation,  manner  of,  52 

of  agar,  62 

of  animals,  84 

of  gelatin,  62 
Insecticides,  235 
Instruments,  sterilization  of,  28 
Intestine,  bacteria  in,  231 
lodin,  31 
Iris  blender,  27 


JAPANESE    method    of    preparing 

agar,  56 
Jenner's  stain,  36,  189 


KLATSCH  preparations,  69 
Klein's  method  of  staining  spores, 

42 
Koch's  alkaline  methylene-blue,  32 

rules  in  regard  to  bacterial  cause 
of  disease,  87 

steam-chest,  46 

Koch-Lb'fBer  boullion-gelatin,   53 
Koch- Weeks  bacillus,  165 
Kiihne's  stain,  33,  42 


LACTOSE,  60 
Leishman's  stain,  35 
Lepra  bacillus,  120 


Leprosy,  120 
Leptothrix  buccalis,  101 
Leukocytes,  washed,  89 
Liborius's    method    of    cultivating 

anaerobic  bacteria,  71 
Life-cycle  of  malarial  sporozoa,  184 

of  protozoa,  184 
Light,    influence    of,    on    bacteria, 

24 

Litmus-agar,  60 
Lb'ffler's    alkaline    methylene-blue, 

32 

blood-serum  mixture,  60 
bouillon,  49 

mordant  for  flagella,  33,  43 
stain  for  bacillus  of  glanders,  122 
Lysins,  81 


MACCONKEY'S  bile-salt  media,  61 
Macrocytases,  80 
Macrogamete,  187 
Macrophages,  80 
Madura  foot,  214 
Malaria,  184 

Malarial     organisms,     method     of 
examination  for,  189 

protozoa,  estivo-autumnal  fcrm, 

187 

quartan  form,  187 
tertian  form,  187 

sporozoa,   life-cycle   of,    184 
Mai  de  pis,  203 
Malignant  edema,  174 

pustule,  105 
Mallein,  123 
Malta  fever,  181 
Marchoux's  thionin  stain,  189 
Mastigophora,  184 
Media,  fluid,  49 

neutralization  of,  49 
Schultz's  method,  50 

nutrient,  49 

solid,  51 

transparent,  53 
Mediterranean  fever,  181 
Merozoites,  185 
Metchnikoff's  theory  of  immunity, 

80 

Methylene-blue,  alkaline,  32 
Microbe  en  huit,  198 


INDEX 


287 


Micrococcus,  17 

cereus  albus,  162 
flavus,  162 

cholera  gallinarum,  198 

citreus  conglomerata,  168 

gonorrhoeae,  166 

indicus,  92 

melitensis,  181 

of  mal  de  pis,  203 

of  sputum  septicemia,  151 

pasteuri,  151 

pyogenes  citreus,  162 
tenius,  162 

subflavus,  169 

tetragenus,  155 

ureae,  103 
Microcytases,  80 
Microgametes,  187 
Microgametocytes,  187 
Microphages,  80 
Microscope,  26 
Microsporon  furfur,  211 
Microtome,  39 
Milk,  bacteria  in,  95,  228 

culture-medium,  61 

examination     of,     for     tubercle 

bacilli,  114 

in  stained  specimen,  98 
Milzbrand  bacillus,  105 
Moist  chambers,  66 
Molds,  206 

examination  of,  212 

true,  209 
Mordants,  31 

Mosquito,  sexual  cycle  in,  187 
Mouse  septicemia,  202 
Mouth,  bacteria  in,  230 
Mucor  mucedo,  209 
Mycetoma,  214 


NAG  AN  A,  191 

Nail  cultures,  150 

Nasal  cavity,  bacteria  in,  231 

Neapolitanus  bacillus,  135 

Negri  bodies,  193 

Neisser's  stain  for  diphtheria,  34 

for  gonococcus,  167 
Neutralization  of  media.  49 

Schultz's  method,  50 
Nicolle's  stain,  34 


Nitrification,  24 

Nitrifying  organisms  in  soil,  227 

Nivellier      leveling      and      cooling 

apparatus,  65 
Nutrient  agar,  preparation,  55 

blood-serum,  preparation,  57 

media,  49 


ODORS  from  bacteria,  25 
Oi'dium,  207 

albicans,  208 

coccidioides,  208 

lactis,  207 

mycosis,  208 
Oil  immersion,  26 
Ophthalmic  tuberculin  reaction  of 

Calmette,  119 
Opsonic  index,  83,  89 

technic,  87 
Opsonins,  82 
Oven,  hot-air,  45 
Oxygen,  influence  of,  on  life  and 

growth  of  bacteria,  23 


PARACOLON  bacillus,  136 
Parasites,  23 
Parasitic  bacteria,  75 
Paratyphoid  bacillus,  136 
Park's      method      of      cultivating 

anaerobic  bacteria,  74 
Pasteur-Chamberland  filter,  221 
Penicillium  glaucum,  209 
Peptone  solution,  Dunham's,  60 
Petri  saucers,  66 

method     of    .collecting    bacteria 

from  air,  218 

Pfeiffer's  cholera  immunity,  143 
Phagocytosis,  method  of  measuring, 

88 

Phagolysis,  80 
Phenol,  235 

solutions,  33 
Phosphorescence,  25 
Phosphorescent  bacteria,  100 
Pigmentation,  25 
Pink-eye,  165 
Piroplasma  bigeminum,  192 

bo  vis,  192 
Plague,  bubonic,  177 


288 


INDEX 


Plate  cultures,  64 
Pneumobacillus,  150,  151 
Pneumococcus,  150 
Pneumonia,  148 
Potassium  iodid  medium,  60 
Potato  mash,  52 
Potato-cultures,  51 
Potato-gelatin,  60 
Potatoes,  test-tube,  52 
Precipitins,  82 
Preservatives,  234 
Proteins,  25 
Proteus  mirabilis,  205 

vulgaris,  205 

zenkeri,  205 
Protozoa,  183 

life-cycle  of,  184 

malarial,    estivo-autumnal    form 

of,  187 

quartan  form  of,  187 
tertian  form  of,  187 
Pseudobacillus  of  Hoffman,  126 
PtomaYns,  24,  77 
Puerperal  fever,  160 
Pustule,  malignant,  105 
Putrefaction,  25 

RAUSCHBRAND,  198 
Ray-fungus,  212 
Relapsing  fever,  193 
Removing  excess  of  stain,  37 
Rhinoscleroma,  153 
Rolled  cultures,  67 
Romanowsky's  stain,  35 
Rb'ntgen    rays,    influence    of,     on 

bacteria,  24 
Root  bacillus,  93 
Rotz-bacillus,  121 
Rouget  du  pore,  201 
Roux's  double  stain,  34 

method  of  cultivating  anaerobic 
bacteria,  73 

test-tube,  52 

SACCHAROLYTIC  bacteria,  139 
Saccharomyces  albicans,  207 

cerevisise,  206 
•  mycoderma,  207 

niger,  207 

rosaceus,  207 


Saccharomycetes,  206 
Salts  of  metals,  234,  235 
Sapremia,  76 
Saprophytes,  23 
Saprophytic  bacteria,  75 
Sarcina,  104 

aurantica,  105 

lutea,  104 

ventriculi,  105 
Sarcodina,  184 
Schizogony,  184 
Schizomycetes,  17 
Schizophycese,  17 
Schizophyta,  17 
Schultz's  method  of  neutralization 

of  media,  50 

Schweinerotlauf bacillus,  201 
Sedgwick-Tucker    method    of    col- 
lecting bacteria  from  air,   219 
Septicemia,  76 

fowl,  198 

hemorrhagic,  200 

mouse,  202 
Serum,  antituberculous,  119 

hemolytic,  81 

Sewage  bacteria,  examination  for, 
224 

bacterial  treatment  of,  226 
Sexual  cycle  in  mosquito,  187 
Skin,  bacteria  on,  229 

examination  of,  229 
Sleeping  sickness,  192 
Small-pox,  196 
Smear  culture,  63 
Smegma  bacillus,  120 
Smith's  fermentation  tube,  62 
Soap,  236 
Soil,  bacteria  in,  227 

examination  of,  227 
Solid  media,  51 

transparent  media,  53 
Soor,  208 
Spatula,  39 
Spirillum,  17,  103 

cholerae,  140 

bacteria  similar  to,  146 

concentricum,  103 

Finkleri,  146 

of  relapsing  fever,  193 

rubrum,  103 

tyrogenum,  147 


INDEX 


289 


Spirochaeta  Obermeieri,  193 

pallida,  194 

Spironema  pallidum,  194 
Splenic  fever,  105 
Sporangium,  210 
Spores,  arthro-,  20 
contents  of,  20 
endo-,  20 
formation  of,  20 

requisites,  21 
resistance  of,  22 
staining  of,  41 

Klein's  method,  42 
Kuhne's  method,  42 
Weigert's  method,  43 
Sporocyte,  185 
Sporogony,  184 
Sporozoa,  184 

malarial,  life-cycle  of,  184 
Stab  cuture,  63 
Stain,  alkaline,  31 
capsule,  43 

Buerger's  method,  43 
flagella,  with  Lb'ffler's  mordant, 

43 
Frankel's,   for  tubercle  bacillus, 

IJ3 

Gabbet's  acid  blue,  33 
Giemsa's,  35 
Gram's  iodin,  33 
Jenner's,  36,  189 
Koch's,  32 
Kuhne's,  33 
Leishman's,  35 
Lb'ffler's,  32 

for  bacillus  of  glanders,  122 

mordant,  33 
Marchoux's,  189 
Neisser's,  34,  167 
Nicolle's,  34 
removing  excess  of,  37 
Romanowsky's,  35 
Roux's  double,  34 
Unna's  methyl-blue,  33 
Wright's,  190 
Ziehl-Neelsen,  33 
Staining,  30 

fungi,  Unna's  method,  43 
general  method,  36 
Gram's  double  method,  40 

method  for  tissues,  41 

19 


Staining  of  spores,  41 
Klein's  method,  42 
Kuhne's  method,  42 
Weigert's  method,  43 

of  tissue  sections,  37,  38 

solutions,  30 

special  methods,  40 

tubercle  bacilli  in  tissue,  116 
Staphylococcus,  18 

pyogenes  albus,  162 

aureus,  160 
Steam  sterilizer,  Arnold's,  47 

superheated,  234 
Steam-chest,  Koch's,  46 
Stegomyia  fasciata,  196 
Sterilization,    fractional,    of    Tyn- 
dall,  47 

of  blood-serum,  58 

of  bouillon,  50 

of  cultures,  44 

of  hands,  236 

of  instruments,  28 
Sterilizer,  Arnold's,  47 
Sterilizing  gelatin,  55 
Stomach,  bacteria  in,  231 
Streptococcus,  18 

erysipelatis,  159 

in  diphtheria,  129 

puerperalis,  160 

pyogenes,  159 
Streptothrices,  212 
Streptothrix  actinomyces,  212 

farcinica,  215 

madurae,  214 
Stroke  culture,  63 
Sulphur  dioxid,  235 
Suppuration,  158 
Suppurative  bacteria,  76 
Susceptibility,  75,  76 

acquired,  76 

inherited,  76 
Syphilis,  194 

bacillus,  1 20 


TEMPERATURE,    influence    of     on 
life  and  growth  of  bacteria,  23 

Test-tube,  48 
cultures,  63 
potatoes,  52 

Tetanus,  170 


INDEX 


Thermoregulators,  59 
Thermostat  for  blood-serum,  58 
Thread  reaction,  82 
Thrush  fungus,  208 
Thrust  culture,  63 
Tick  fever,  African,  194 
Tissue  preparations,  38 
Torula  cerevisise,  206 
Toxalbumins,  25,  77 
Toxins,  25 

nature  of,  77 

of  diphtheria,  128 
Toxone  of  diphtheria,  128 
Treponema  pallidum,  194 
Trichophyton  tonsurans,  210 
Trypanosoma  Brucei,  191 

Castellani,  192 

equiperdum,  192 

Evansi,  192 

hominis,  192 

Lewisi,  191 

neprevi,  192 

Rougetii,  192 

ugandense  gambiense,  192 
Trypanosomes,  190 
Trypanosomiasis,  192 
Tsetse-fly  disease,  191 
Tubercle  bacillus,  109 

products  of,  118 
Tuberculin,  Deny's  B.  F.,  119 

R,  118 

reaction,    ophthalmic,     of     Cal- 

mette,  119 
Tuberculocidin,  118 
Tuberculosis,  109 
Tyndall,  fractional  sterilization   of, 

47 
Typhoid  fever,  129 

medium,  Eisner's,  60 
of  Hiss,  6 1 

fever,  Widal  reaction  in,  131 
Typhotoxin,  136 


UNNA'S  borax  methyl-blue,  33 
method  of  staining  fungi,  43 

Urethra,  bacteria  in,  168 

Urine,  bacteria  in,  102 
media  for  gonococci,  61 

Urobacillus  liquefaciens,  103 


VACCINES,  protective,  in  pus  infec- 
.  tions,  170 
Vaccinia,  196 
Vagina,  bacteria  in,  168 
Variola,  196 
Vibrio  Metchnikovi,  147 
Vibrion  butyrique  of  Pasteur,  97 

septicemia,  148 

septique,  174 


WASHED  leukocytes,  89 
Water,  artesian  well,  221 

bacteria  in,  220,  223 

boiling  of,  222 

cholera  bacillus  in,  143 

examination  of,  220,  223 

filtered,  221 

non-pathogenic  bacteria  in,  99 

purity  of,  220 

typhoid  bacillus  in,  135 
Weak  solutions,  32 
Weigert's  method  of  staining  spores, 

43 

Welch's  capsule  stain,  35 
Wertheim's  method  of  cultivating 

gonococcus,  1 66 
White  mouth,  208 
Widal  reaction  in  typhoid  fever, 

J31 

Wire  cage,  48 
WolfhiigePs  apparatus,  225 
Wright's  blood-capsule,  89 

chromatin  stain,  190 

method  of  cultivating  anaerobic- 
bacteria,  74 
Wurzel  bacillus,  93 


YEASTS,  206 

examination  of,  212 

pathogenic,  207 
Yellow  fever,  196 
Yokote's  method  of  preparing 

agar,  56 


ZIEHL-NEELSEN  stain,  33 
Zooglea,  1 8 


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practical.  It  is  in  the  fullest  sense  an  applied  anatomy — an  anatomy  that  will 
be  of  inestimable  value  to  the  surgeon  because  only  those  facts  are  discussed 
and  only  those  structures  and  regions  emphasized  that  have  a  peculiar  interest 
to  him.  Dr.  Campbell  has  treated  his  subject  in  a  very  systematic  way,  and 
illustrated  his  descriptions  so  graphically  that  the  acquisition  of  anatomic  rela- 
tions has  been  rendered  much  more  easy  than  has  ever  before  been  attempted. 


SURGERY   AND    ANATOMY. 


Fowler's 
Treatise  on    Surgery 

A  Treatise  on  Surgery.  By  GEORGE  R.  FOWLER,  M.  D., 
Emeritus  Professor  of  Surgery,  New  York  Polyclinic.  Two 
imperial  octavos  of  725  pages  each,  with  888  original  text-illus- 
trations and  4  colored  plates.  Per  set:  Cloth,  $15.00  net; 
Half  Morocco,  $18.00  net. 

RECENTLY  ISSUED— IN  TWO  VOLUMES 

Without  doubt,  Dr.  Fowler's  work  is  the  most  practical  and  complete  sur- 
gery for  its  size  ever  published.  Every  sentence  fells  its  story,  either  to  re- 
count a  fact  or  give  instruction  as  to  treatment.  The  author  especially  em- 
phasizes those  injuries  and  surgical  diseases  that  are  of  the  greatest  import- 
ance, not  only  because  of  their  frequency,  but  also  because  of  the  difficulty 
of  diagnosis  and  the  special  care  demanded  in  their  treatment.  The  text  is 
elaborately  illustrated  with  888  entirely  new  and  original  illustrations,  and 
every  picture  actually  shows  some  surgical  procedure,  some  step  in  the  technic 
of  an  operation  ;  every  picture  indicates  precisely  how  to  do  something. 

Rudolph    Matas,    M.  D.,  Professor  of  Surgery,  Tulane  University  of  Louisiana. 

"  After  a  careful  examination  of  this  work  I  am  glad  to  state  that  the  completed  text 
fully  confirms  the  assurance  I  entertained  :  that  this  would  prove  a  work  of  high  order  and 
distinct  merit.  These  splendid  volumes  fully  justify  the  repute  of  their  author  for  earnest- 
ness, thoroughness,  and  learning." 


Gant  on  Constipation  and 
Intestinal  Obstruction 


Constipation  and  Intestinal  Obstruction.  By  SAMUEL 
G.  GANT,  M.  D.,  Professor  of  Diseases  of  the  Rectum  and  Anus, 
New  York  Post-Graduate  Medical  School  and  Hospital.  Octavo 
of  600  pages,  with  235  original  illustrations. 

READY    IN    SEPTEMBER 

This  is  a  work  for  every  practitioner  and  surgeon.  The  consideration 
given  to  the  medical  treatment  of  constipation  is  unusually  extensive,  and 
the  chapter  devoted  to  formulas  will  also  be  found  invaluable  to  the  practi- 
tioner. The  descriptions  of  the  operative  procedures  are  concise,  yet  fully 
explicit.  The  235  original  pictures  are  as  practical  as  the  text. 


SAUNDERS'  BOOKS  ON 


Moynihan's 
Abdominal  Operations 


Abdominal  Operations.  By  B.  G.  A.  MOYNIHAN,  M.  S. 
(LOND.),  F.R.C.S.,  England.  Octavo,  beautifully  illustrated. 
Cloth,  $7.00  net;  Half  Morocco,  $8.50  net. 

RECENTLY    ISSUED-NEW    (2d)    EDITION 
TWO   LARGE   EDITIONS    IN   ONE  YEAR 

It  has  been  said  of  Mr.  Moynihan  that  in  describing  details  of  operation* 
he  is  at  his  best.  The  appearance  of  this,  his  latest  work,  therefore,  will  ba 
widely  welcomed  by  the  medical  profession,  giving,  as  it  does,  in  most  cleai 
and  exact  language,  not  only  the  actual  modus  operanat  of  the  various  ab- 
dominal operations,  but  also  the  preliminary  technic  of  preparation  and 
sterilization. 

Edward  Martin,  M.  D. 

Professor  of  Clinical  Surgery,  University  of  Pennsylvania 

"  It  is  a  wonderfully  good  book.  He  has  achieved  complete  success  in  illustrating,  both 
by  words  and  pictures,  the  best  technic  of  the  abdominal  operations  now  commonly 
performed." 


Moynihan  on  Gall-stones 


Gall-stones  and  their  Surgical  Treatment.    By  B.  G.  A. 

MOYNIHAN,  M.S.  (LOND.),  F.R.C.S.,  Senior  Assistant  Surgeon, 
Leeds  General  Imfirmary,  England.  Octavo  of  458  pages,  fully 
illustrated.  Cloth,  $5.00  net ;  Half  Morocco,  $6.50  net. 

RECENTLY  ISSUED— NEW  (2d)  EDITION 

Mr.  Moynihan,  in  revising  his  book,  has  made  many  additions  to  the  text, 
so  as  to  include  the  most  recent  advances.  Especial  attention  has  been  given 
to  a  detailed  description  of  the  early  symptoms  in  cholelithiasis,  enabling  a 
diagnosis  to  be  made  in  the  stage  in  which  surgical  treatment  can  be  most 
safely  adopted.  A  number  of  the  illustrations  are  in  color. 

British  Medical  Journal 

"  He  expresses  his  views  with  admirable  clearness,  and  he  supports  them  by  a  large 
number  of  clinical  examples,  which  will  be  much  prized  by  those  who  know  the  difficult 
problems  and  tasks  which  gall-stone  surgery  not  infrequently  presents." 


SURGER  Y  AND  ANA  TOMY 


Scudder's  Fractures 

WITH   NOTES   ON   DISLOCATIONS 


The  Treatment  of  Fractures :  with  Notes  on  a  few  Com- 
mon Dislocations.  By  CHARLES  L.  SCUDDER,  M.  D.,  Surgeon  to 
the  Massachusetts  General  Hospital,  Boston.  Octavo  volume  of 
660  pages,  with  854  illustrations.  Polished  Buckram,  $5.50  net; 
Half  Morocco,  $7.00  net. 

JUST    ISSUED— NEW    (6th)    EDITION,    ENLARGED 
OVER    25,000    COPIES 

Six  large  editions  of  this  remarkable  book  is  an  undoubted  indication  of 
the  popularity  of  Dr.  Scudder's  work.  For  this  new  edition  numerous  addi- 
tions have  been  made  throughout  the  text  and  a  large  number  of  new  illustra- 
tions added,  greatly  enhancing  the  value  of  the  work.  The  articles  on 
Dislocations,  illustrated  in  that  practical  manner  which  has  made  Dr. 
Scudder's  book  so  useful,  will  be  found  extremely  valuable. 

Joseph  D.  Bryant,  M.D.,   Professor  of  the  Principles  and  Practice  of  Surgery* 

University  and  Bellevue  Hospital  Medical  College. 

"As  a  practical  demonstration  of  the  topic  it  is  excellent,  and  as  an  example 
of  bookmaking  it  is  highly  commendable." 


Bickham's  Operative  Surgery 

A  Text-Book  of  Operative  Surgery.  By  WARREN  STONE 
BICKHAM,  M.  D.,  PHAR.  M.,  of  New  York  City.  Octavo  of  1000 
pages,  with  559  illustrations.  Cloth,  $6.co  net;  Half  Morocco, 
$7.50  net. 

RECENTLY    ISSUED— NEW    (2d)    EDITION 

This  absolutely  new  work  completely  covers  the  surgical  anatomy  and 
operative  technic  involved  in  the  operations  of  general  surgery.  The  practica- 
bility of  the  work  is  particularly  emphasized  in  the  559  magnificent  illustrations. 

Boston  Medical  and  Surgical  Journal 

"  The  book  is  a  valuable  contribution  to  the  literature  of  operative  surgery.  It  repre- 
sents a  vast  amount  of  careful  work  and  technical  knowledge  on  the  part  of  the  author. 
For  the  surgeon  in  active  practice  or  the  instructor  of  surgery  it  is  an  unusually  good  review 
of  the  subject." 


SAUNDERS'    BOOKS    ON 


Gould's  Operations  on  the 
Intestines    and    Stomach 


The  Technic  of  Operations  upon  the  Intestines  and 
Stomach.  By  ALFRED  H.  GOULD,  M.  D.,  of  Boston,  Massa- 
chusetts. Large  octavo,  with  190  original  illustrations.  Cloth, 
$5.00  net;  Half  Morocco,  $6.50  net. 

RECENTLY    ISSUED 

Dr.  Gould's  new  work  is  the  result  of  exhaustive  experimentation,  the 
technic  of  the  operations  described  being  simplified  as  far  as  possible  by  ex- 
periments on  animals,  thus  leading  to  the  development  of  many  new  features. 
The  text  is  purposely  concise,  the  technic  being  presented  very  clearly  by  the 
numerous  practical  illustrations. 

New  York  State  Journal  of  Medicine 

"  The  illustrations  are  so  good  that  one  scarcely  needs  the  text  to  elucidate  the  steps  of 
the  operations  described.  The  work  represents  the  best  surgical  knowledge  and  skill." 


DaCosta's  Modern  Surgery 

Modern  Surgery.  GENERAL  AND  OPERATIVE.  By  JOHN 
CHALMERS  DACOSTA,  M.  D.,  Professor  of  the  Principles  of  Sur- 
gery and  of  Clinical  Surgery  in  the  Jefferson  Medical  College, 
Philadelphia.  Octavo  of  1283  pages,  with  872  illustrations. 
Cloth,  $5.50  net;  Half  Morocco,  $7.00  net. 

RECENTLY   ISSUED— THE   NEW  (5th)    EDITION 

For  this  new  fifth  edition  the  book  has  undergone  a  thorough  and  careful 
revision,  and  there  have  been  added  much  new  matter  and  nearly  two  hundred 
excellent  and  practical  illustrations.  Many  new  subjects  and  operations  have 
been  incorporated. 

Boston  Medical  and  Surgical    Journal 

*'  We  commend  the  book,  as  we  have  previously  commended  it,  to  surgeons  and  to 
students  as  the  most  satisfactory  one-volume  contemporaneous  treatise  on  surgery  pub- 
lished in  this  country." 


SURGERY  AND    ANATOMY. 


Schultze  and  Stewart's 
Topographic  Anatomy 

Atlas  and  Text=Book  of  Topographic  and  Applied 
Anatomy.  By  PROF.  DR.  O.  SCHULTZE,  of  Wiirzburg.  Edited, 
with  additions,  by  GEORGE  D.  STEWART,  M.D.,  Professor  of 
Anatomy  and  Clinical  Surgery,  University  and  Bellevue  Hospital 
Medical  College,  New  York.  Large  quarto  of  187  pages,  with 
25  colored  figures  on  22  colored  lithographic  plates,  and  89  text- 
cuts,  60  in  colors.  Cloth,  $5.50  net. 

RECENTLY   ISSUED 

It  was  Professor  Schultze' s  special  aim  to  produce  a  text-book  and  atlas 
not  for  the  anatomist  alone,  but  more  particularly  for  the  general  practitioner. 
The  value  of  the  knowledge  of  topographic  anatomy  in  bedside  diagnosis  is 
emphasized  throughout  the  book. 

Arthur  Dean  Bevan,  M.  D. 

"  I  regard  it  as  a  very  admirable  work,  for  students  especially,  and  find  the  plates  and 
the  text  excellent." 

Sobotta  and  McMurrich's 
Human  Anatomy 

Atlas  and  Text-Book  of  Human  Anatomy.  In  Three 
Volumes.  By  J.  SOBOTTA,  M.  D.,  of  Wiirzburg.  Edited,  with 
additions,  by  J.  PLAYFAIR  McMuRRiCH,  A.  M.,  PH.  D.,  Professor 
of  Anatomy,  University  of  Michigan.  Three  large  quartos,  each 
containing  250  pages  of  text  and  over  300  illustrations,  mostly  in 
colors.  Per  volume  :  Cloth,  $6.00  net. 

VOLUME    III    JUST    READY— COMPLETING    THE    WORK 
Edward  Martin,   M.D.,  University  of  Pennsylvania. 

"  This  is  a  piece  of  bookmaking  which  is  truly  admirable,  with  plates  and  text  so  well 
chosen  and  so  clear  that  the  work  is  most  useful." 


10  SAUNDERS  BOOKS  ON 

Eisendrath's 
Surgical  Diagnosis 

A  Text-Book  of  Surgical  Diagnosis.  By  DANIEL  N.  EISEN- 
DRATH,  M.  D.,  Adjunct  Professor  of  Surgery  in  the  College  of 
Physicians  and  Surgeons,  Chicago.  Octavo  of  775  pages,  with 
482  entirely  new  and  original  text-illustrations  and  some  colored 
plates.  Cloth,  $6.50  net;  Half  Morocco,  $8.00  net. 

RECENTLY    ISSUED 
WITH    482    ORIGINAL    ILLUSTRATIONS 

Dr.  Eisendrath  takes  up  each  disease  and  injury  amenable  to  surgical 
treatment,  and  sets  forth  the  means  of  correct  diagnosis  in  a  systematic  and 
comprehensive  way.  Definite  directions  as  to  methods  of  examination  are  pre- 
sented clearly  and  concisely,  providing  for  all  contingencies  that  might  arise 
in  any  given  case.  Each  one  of  the  four  hundred  and  eighty-two  magnifi- 
cent illustrations  indicates  precisely  how  to  diagnose  the  condition  considered. 

Surgery,  Gynecology,  and  Obstetrics 

"  The  book  is  one  which  is  well  adapted  to  the  uses  of  the  practising  surgeon  who 
desires  information  concisely  and  accurately  given." 

Eisendrath's  Clinical  Anatomy 

A  Text-Book  of  Clinical  Anatomy.  By  DANIEL  N.  EISEN- 
DRATH, A.  B.,  M.  D.,  Adjunct  Professor  of  Surgery  in  the  Col- 
lege of  Physicians  and  Surgeons,  Chicago.  Octavo  of  535  pages, 
with  original  illustrations.  Cloth,  $5.00  net. 

RECENTLY    ISSUED— NEW    (2d)    EDITION 

This  new  anatomy  discusses  the  subject  from  the  clinical  standpoint.     A 
portion  of  each   chapter  is  devoted  to  the  examination  of  the  living  through 
palpation  and  marking  of  surface  outline  of  landmarks,  etc.     The  illustrations 
are  original. 
Medical  Record,  New  York 

"  A  special  recommendation  for  the  figures  is  that  they  are  mostly  original  and  were 
made  for  the  purpose  in  view.  The  sections  of  joints  and  trunks  are  those  of  formalinized 
cadavers  and  are  unimpeachable  in  accuracy." 


SURGE R  Y  AND  ANA  TO  MY 


International 
Text-Book  of  Surgery 

Second  Edition,  Thoroughly  Revised  and  Enlarged 


The  International  Text-Book  of  Surgery.  In  two  vol- 
umes. By  American  and  British  authors.  Edited  by  J.  COLLINS 
WARREN,  M.  D.,  LL.  D.,  F.  R.  C.  S.  (Hon.),  Professor  of  Sur- 
gery, Harvard  Medical  School;  and  A.  PEARCE  GOULD,  M.  S., 
F.  R.  C.  S.,  of  London,  England.  —  Vol.  I.  General  and  Opera- 
tive Surgery.  Royal  octavo,  975  pages,  461  illustrations,  9  full- 
page  colored  plates.  —  Vol.  II.  Special  or  Regional  Surgery. 
Royal  octavo,  1122  pages,  499  illustrations,  and  8  full-page 
colored  plates.  Per  volume  :  Cloth,  $5.00  net. 

American  TEXT-BOOK  OF  Surgery 

FOURTH  EDITION 

American  Text-Book  of  Surgery.  Edited  by  W.W.  KEEN, 
M.  D.,  LL.  D.,  HON.  F.  R.  C.  S.,  ENG.  AND  EDIN.;  and  J.WILLIAM 
WHITE,  M.  D.,  PH.  D.  Octavo,  1363  pages,  551  text-cuts  and  39 
colored  and  half-tone  plates.  Cloth,  $7.00  net. 


Robson  &  Cammidg'e  on  Pancreas 

The  Pancreas  :    its  Surgery  and   Pathology.     By  A.  W. 

MAYO  ROBSON,  F.  R.  C.  S.,  of  Leeds,  England;  and  P.  J.  CAM- 

MIDGE,  F.  R.  C.  S.,  of  London,  England.     Octavo  of  546  pages, 

illustrated.  Cloth,  $5.00  net. 

JUST  READY 

This  new  work,  upon  one  of  the  most  widely  discussed  subjects  of  the 
times,  represents  the  original  investigations  of  these  eminent  authorities.  It 
takes  up  Anatomy,  Embryology,  Histology,  Physiology,  Pathology,  Symptom- 
atology, and  Injuries  and  Diseases,  and  there  are  special  chapters  on  Chemi- 
cal Pathology  and  Diabetes.  The  text  is  illustrated. 


12  SAUNDER&  BOOKS  ON 


American  Illustrated  Dictionary 

THE  AMERICAN  ILLUSTRATED  MEDICAL  DICTIONARY.  With 
tables  of  Arteries,  Muscles,  Nerves,  Veins,  etc.  ;  of  Bacilli, 
Bacteria,  etc.  ;  Eponymic  Tables  of  Diseases,  Operations, 
Stains,  Tests,  etc.  By  W.  A.  NEWMAN  DORLAND,  M.  D. 
Large  octavo,  840  pages.  Flexible  leather,  $4.50  net;  with 
thumb  index,  $5.00  net. 

Howard    A.    Kelly,  M.  D.,    Professor  of   Gynecology,  Johns  Hopkins 
"Dr.  Borland's  dictionary  is  admirable.     It  is  so  well  gotten   up  and  of 
such  convenient  size.     No  errors  have  been  found  in  my  use  of  it." 

Golebiewski  and  Bailey's  Accident  Diseases 

ATLAS  AND  EPITOME  OF  DISEASES  CAUSED  BY  ACCIDENTS. 
By  DR.  ED.  GOLEBIEWSKI,  of  Berlin.  Edited,  with  additions, 
by  PEARCE.  BAILEY,  M.D.  Cloth,  $4.00  net.  In  Sounders' 
Hand-  Atlas  Series. 

Helferich  and  Blood  good  on  Fractures 

ATLAS  AND  EPITOME  OF  TRAUMATIC  FRACTURES  AND  DISLO- 
CATIONS. By  PROF.  DR.  H.  HELFERICH,  of  Greifswald,  Prussia. 
Edited,  with  additions,  by  JOSEPH  C.  BLOODGOOD,  M.D.,  Asso- 
ciate in  Surgery,  Johns  Hopkins  University,  Baltimore.  216 
colored  figures  on  64  lithographic  plates,  190  text-cuts,  and 
353  Pages  of  text.  Cloth,  $3.00  net.  In  Sounders'  Atlas  Series. 

Sultan  and  Coley  on  Abdominal  Hernias 

ATLAS  AND  EPITOME  OF  ABDOMINAL  HERNIAS.  By  PR.  DR. 
G.  SULTAN,  of  Gottingen.  Edited,  with  additions,  by  WM. 
B.  Coley,  M.D.  Cloth,  $3.00  net.  In  Sounders'  Hand-  Atlas 
Series. 

Warren's  Surgical  Pathology  |edc^ 

SURGICAL  PATHOLOGY  AND  THERAPEUTICS.  By  J.  COLLINS 
WARREN,  M.D.,  LL.D.,  F.R.C.S.(HoN.),  Professor  of  Sur- 
gery, Harvard  Medical  School.  Octavo,  873  pages,  136  illus- 
trations. Cloth,  $5.00  net  ;  Half  Morocco,  $6.50  net. 

Zuckerkandl  and  DaCosta's  Surgery  f^ 

ATLAS  AND  EPITOME  OF  OPERATIVE  SURGERY.  By  DR.  O. 
ZUCKERKANDL,  of  Vienna.  Edited,  with  additions,  by  J. 
CHALMERS  DACOSTA,  M.D.,  Professor  of  the  Principles  of  Sur- 
gery and  Clinical  Surgery,  Jefferson  Medical  College,  Phila. 
40  colored  plates,  278  text-cuts,  and  410  pages  of  text. 
Cloth,  $3.50  net.  In  Sounders'  Atlas  Series. 


SURGERY  AND  ANATOMY.  13 

Lewis'  Anatomy  and  Physiology  for  Nurses 

Recently  Issued 

ANATOMY  AND  PHYSIOLOGY  FOR  NURSES.  By  LEROY  LEWIS,  M.D., 
Surgeon  to  and  Lecturer  on  Anatomy  and  Physiology  for  Nurses  at  the 
Lewis  Hospital,  Bay  City,  Michigan.  I2mo  of  317  pages,  with  146  il- 
lustrations. Cloth,  $1.75  net. 

A  demand  for  such  a  work  as  this,  treating  tJie  subjects  from  the  nurse' s point  of 
•view,  has  long-  existed.  Dr.  Lewis  has  based  the  plan  and  scope  of  this  work  on  the 
methods  employed  by  him  in  teaching  these  branches,  making  the  text  unusually 
simple  and  clear. 

"  It  is  not  in  any  sense  rudimentary,  but  comprehensive  in  its  treatment  of  the 
subjects  in  hand." — Nurses  Journal  of  the  Pacific  Coast, 

McClellan's  Art  Anatomy  Recently  issued 

ANATOMY  IN  ITS  RELATION  TO  ART.  By  GEORGE  MCCLELLAN,  M.D., 
Professor  of  Anatomy,  Pennsylvania  Academy  of  the  Fine  Arts.  Quarto 
volume,  9  by  12*^  inches,  with  338  original  drawings  and  photographs, 
and  260  pages  of  text.  Dark  blue  vellum,  $10.00  net;  Half  Russia, 
$12.50  net. 

Senn    On    Tumors  Second  Revised  Edition 

PATHOLOGY  AND  SURGICAL  TREATMENT  OF  TUMORS.  By  NICHOLAS 
SENN,  M.D.,  PH.D.,  LL.D.,  Professor  of  Surgery,  Rush  Medical  Col- 
lege, Chicago.  Handsome  octavo,  718  pages,  with  478  engravings, 
including  12  full-page  colored  plates.  Cloth,  $5.00  net  ;  Sheep  or  Half 
Morocco,  $6.50  net. 

Macdonald's  Diagnosis  and  Treatment 

A  CLINICAL  TEXT-BOOK  OF  SURGICAL  DIAGNOSIS  AND  TREATMENT, 
ByJ.  W.  MACDONALD,  M.D.  Edin.,  F.R.C.S.  Edin.,  Professor  Emer- 
itus of  the  Practice  of  Surgery  and  of  Clinical  Surgery  in  Hamline  Uni- 
versity in  Minneapolis,  Minn.  Octavo,  798  pages,  handsomely  illustrated. 
Cloth,  $5.00  net ;  Sheep  or  Half  Morocco,  $6.50  net. 

Preiswerk  and  Warren's  Dentistry    Recently  issued 

ATLAS  AND  EPITOME  OF  DENTISTRY.  By  PROF.  G.  PREISWERK,  of 
Basil.  Edited,  with  additions,  by  GEORGE  W.  WARREN,  D.D.S.,  Professor 
of  Operative  Dentistry,  Pennsylvania  College  of  Dental  Surgery,  Philadel- 
phia. With  44  lithographic  plates,  152  text-cuts,  and  375  pages  of  text. 
In  Sounders'  Atlas  Series,  Cloth,  $3.50  net. 

"  Nowhere  in  dental  literature  have  we  ever  seen  illustrations  which  can  begin  to 
compare  with  the  exquisite  colored  plates  produced  in  this  volume." — Dental  Review, 


14  SAUNDERS^    BOOKS   ON 

Haynes'  Anatomy 

A  MANUAL  OF  ANATOMY.  By  IRVING  S.  HAYNES,  M.  D.,  Professor 
of  Practical  Anatomy,  Cornell  University  Medical  College.  Octavo,  680 
pages,  with  42  diagrams  and  134  full-page  half-tones.  Cioth,  $2.50  net. 

American  Pocket  Dictionary 

THE  AMERICAN  POCKET  MEDICAL  DICTIONARY.  Edited  by  W.  A. 
NEWMAN  BORLAND,  A.  M.,  M.  D.,  Assistant  Obstetrician,  Hospital  of 
the  University  of  Pennsylvania,  etc.  578  pages.  Full  leather,  limp, 
with  gold  edges,  $l.oo  net  ;  with  patent  thumb-index,  $1.25  net. 

"  I  am  struck  at  once  with  admiration  at  the  compact  size  and  attractive  exterior. 
I  can  recommend  it  to  our  students  without  reserve."  —  JAMES  W.  HOLLAND,  M.  D., 
Professor  of  Medical  Chemistry  and  Toxicology,  at  the  Jefferson  Medical  Col- 
lege ,  Philadelphia. 

Beck's  Fractures 

FRACTURES.  By  CARL  BECK,  M.  D.,  Professor  of  Surgery,  New 
York  Post-graduate  Medical  School  and  Hospital.  With  an  Appendix 
on  the  Practical  Use  of  the  Rontgen  Rays.  335  pages,  170  illustrations. 
Cloth,  $3.50  net. 

Barton  and  Wells'  Medical  Thesaurus     ReiseSued 

A  THESAURUS  OF  MEDICAL  WORDS  AND  PHRASES.  By  W.  M.  BARTON, 
A.  M.,  M.  D.,  Assistant  to  Professor  of  Materia  Medica  and  Therapeutics, 
Georgetown  University,  Washington,  D.  C.  ;  and  WALTER  A.  WELLS, 
M.  D.,  Demonstrator  of  Laryngology,  Georgetown  University,  Washing- 
ton, D.  C.  I2mo  of  534  pages.  Flexible  leather,  $2.50  net  ;  thumb 
index,  #3.00  net. 


Stoney's  Surgical  Technic 

BACTERIOLOGY  AND  SURGICAL  TECHNIC  FOR  NURSES.  By  EMILY  M. 
A.  STONEY,  Superintendent  at  the  Carney  Hospital,  South  Boston,  Mass. 
Revised  by  FREDERIC  R.  GRIFFITH,  M.D.,  Surgeon,  of  New  York. 
I2mo,  278  pages,  illustrated.  $1.50  net. 

"  These  subjects  are  treated  most  accurately  and  up  to  date,  without  the  super- 
fluous reading  which  is  so  often  employed.  .  .  .  Nurses  will  find  this  book  of  the 
greatest  value  "  —  Trained  Nurse  and  Hospital  Review. 

Grant  on  Face,  Mouth,  and  Jaws 

A  TEXT-BOOK  OF  THE  SURGICAL  PRINCIPLES  AND  SURGICAL  DIS- 
EASES OF  THE  FACE,  MOUTH,  AND  JAWS.  For  Dental  Students.  By 
H.  HORACE  GRANT,  A.  M.,  M.  D.,  Professor  of  Surgery  and  of  Clinical 
Surgery,  Hospital  College  of  Medicine.  Octavo  of  231  pages,  with  68 
illustrations.  Cloth,  $2.50  net. 

"  The  language  of  the  book  is  simple  and  clear.  ,  .  .  We  recommend  the  work  to 
those  for  whom  it  is  intended."  —  Philadelphia  Medical  Journal. 


SURGERY  AND  ANATOMY  15 

Senn's  Surgery 

PRACTICAL  SURGERY.  By  N.  SENN,  M.  D.,  PH.  D.,  LL.  D.,  Pro- 
fessor of  Surgery  in  Rush  Medical  College.  Octavo  of  1133  pages,  with 
(650  illustrations,  many  in  colors.  Cloth,  $6:Oo  net  ;  Half  Morocco, 
•'$7.50  net.  Subscription. 

"A  record  of  the  matured  opinions  and  practice  of  an  accomplished  and  exper- 
ienced surgeon." — Annals  of  Surgery. 

Beck's  Surgical  Asepsis 

A  MANUAL  OF  SURGICAL  ASEPSIS.  By  CARL  BECK,  M.  D.,  Pro- 
fessor of. Surgery,  New  York  Post-graduate  Medical  School  and  Hos- 
pital. 306  pages  ;  65  text-illustrations  and  12  full-page  plates.  Cloth, 
$1.25  net. 

Griffith's  Hand -Book  of  Surgery  Recently  issued 

A  MANUAL  OF  SURGERY.  By  FREDERIC  R.  GRIFFITH,  M.  D.,  Sur- 
geon to  the  Bellevue  Dispensary,  New  York  City.  I2mo  of  579  pages, 
with  417  illustrations.  Flexible  leather,  $2.00  net. 

"Well  adapted  to  the  needs  of  the  student  and  to  the  busy  practitioner  for  a  hasty 
review  of  important  points  in  surgery." — American  Medicine. 

Keen's  Addresses  and  Other  Papers         Recently  issued 

ADDRESSES  AND  OTHER  PAPERS.  Delivered  by  WILLIAM  W.  KEEN, 
M.  D.,  LL.D.,  F.  R.  C.  S.  (Hon.),  Professor  of  the  Principles  of  Surgery 
and  of  Clinical  Surgery.  Jefferson  Medical  College,  Philadelphia.  Octavo 
volume  of  441  pages,  illustrated.  Cloth,  $3-75  net- 

Senn's  Syllabus  of  Surgery 

A  SYLLABUS  OF  LECTURES  ON  THE  PRACTICE  OF  SURGERY.  Ar- 
ranged in  conformity  with  "  American  Text-Book  of  Surgery."  By 
NICHOLAS  SENN,  M.  D.,  PH.  D.,  LL.  D.,  Professor  of  Surgery,  Rush 
Medical  College,  Chicago.  Cloth,  $1.50  net. 

"  The  author  has  evidently  spared  no  pains  in  making  his  Syllabus  thoroughly  com- 
prehensive, and  has  added  new  matter  and  alluded  to  the  most  recent  authors  and 
operations.  Full  references  are  also  given  to  all  requisite  details  of  surgical  anatomy 
and  pathology." — British  Medical  Journal. 

Keen  on  the  Surgery  of  Typhoid 

THE  SURGICAL  COMPLICATIONS  AND  SEQUELS  OF  TYPHOID  FEVER. 
By  WM.  W.  KEEN,  M.  D.,  LL.D.,  F.  R.  C.  S.  (Hon.),  Professor  of 
the  Principles  of  Surgery  and  of  Clinical  Surgery,  Jefferson  Medical 
College,  Philadelphia,  etc.  Octavo  volume  of  386  pages,  illustrated. 
Cloth,  $3.00  net. 

"  Every  surgical  incident  which  can  occur  during  or  after  typhoid  fever  is  amply 
discussed  and  fully  illustrated  by  cases.  .  .  .  The  book  will  be  useful  both  to  the 
surgeon  and  physician." — The  Practitioner,  London. 


16  SURGERY  AND   ANATOMY 

Moore's  Orthopedic  Surgery 

A  MANUAL  OF  ORTHOPEDIC  SURGERY.  By  JAMES  E.  MOORE,  M.  D., 
Professor  of  Clinical  Surgery,  University  of  Minnesota,  College  of  Medi- 
cine and  Surgery.  Octavo  of  356  pages,  handsomely  illustrated.  Cloth, 
$2.50  net. 

"  The  book  is  eminently  practical.  It  is  a  safe  guide  in  the  understanding  and  treat- 
ment of  orthopedic  cases.  Should  be  owned  by  every  surgeon  and  practitioner."  — 
Annals  of  Surgery. 


Fowler's   Operating  Room 

THE  OPERATING  ROOM  AND  THE  PATIENT.  By  RUSSELL  S.  FOWLER, 
M.  D.,  Surgeon  to  the  German  Hospital,  Brooklyn,  New  York.  Octavo 
of  284  pages,  illustrated.  Cloth,  $2.00  net. 

Dr.  Fowler  has  written  his  book  for  surgeons,  nurses  assisting  at  an  operation,  internes, 
and  all  others  whose  duties  bring  them  into  the  operating  room.  It  contains  explicit 
directions  for  the  preparation  of  material,  instruments  needed,  position  of  patient,  etc., 
all  beautifully  illustrated. 

Recently   Issued 

Nancrede's  Principles  of  Surgery  New  (2di  Edition 

LECTURES  ON  THE  PRINCIPLES  OF  SURGERY.  By  CHARLES  B.  NAN- 
CREDE,  M.  D.,  LL.  D.,  Professor  of  Surgery  and  of  Clinical  Surgery, 
University  of  Michigan,  Ann  Arbor.  Octavo,  407  pages,  illustrated. 
Cloth,  $2.50  net. 

"  We  can  strongly  recommend  this  book  to  all  students  and  those  who  would  see 
something  of  the  scientific  foundation  upon  which  the  art  of  surgery  is  built."  — 
Quarterly  Medical  Journal,  Sheffield,  England. 

Recently  Issued 

Nancrede's  Essentials  of  Anatomy.  ?th  Edition 

ESSENTIALS  OF  ANATOMY,  including  the  Anatomy  of  the  Viscera. 
By  CHARLES  B.  NANCREDE,  M.  D.,  Professor  of  Surgery  and  of  Clinical 
Surgery,  University  of  Michigan,  Ann  Arbor.  Crown  octavo,  388  pages, 
1  80  cuts.  With  an  Appendix  containing  over  60  illustrations  of  the 
osteology  of  the  body.  Based  on  Gray's  Anatomy.  Cloth,  $1.00  net. 
In  Saunders*  Question  Compends. 

"  The  questions  have  been  wisely  selected,  and  the  answers  accurately  and  con- 
cisely given."  —  University  Medical  Magazine. 

Martin's   Essentials   Of  Surgery.      Seventh  Revised  Edition 

ESSENTIALS  OF  SURGERY.  Containing  also  Venereal  Diseases,  Surgi- 
cal Landmarks,  Minor  and  Operative  Surgery,  and  a  complete  description, 
with  illustrations,  of  the  Handkerchief  and  Roller  Bandages.  By  ED- 
WARD MARTIN,  A.  M.,  M.  D.,  Professor  of  Clinical  Surgery,  University 
of  Pennsylvania,  etc.  Crown  octavo,  338  pages,  illustrated.  With  an 
Appendix  on  Antiseptic  Surgery,  etc.  Cloth,  $1.00  net.  In  Saunders1 
Question  Compends. 

Martin's   Essentials  of  Minor  Surgery,   Bandaging, 
and  Venereal  Diseases.     Second  Revised  Edition 

ESSENTIALS  OF  MINOR  SURGERY,  BANDAGING,  AND  VENEREAL  DIS- 
EASES. By  EDWARD  MARTIN,  A.  M.,  M.  D.,  Professor  of  Clinical  Sur- 
gery, University  of  Pennsylvania,  etc.  Crown  octavo,  1  66  pages,  with 
78  illustrations.  Cloth,  $1.00  net.  In  Sounders'  Question  Compends. 


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10.  ESSENTIALS  OF  GYNECOLOGY.    6th  ed.    With  57  illustrations. 

By  EDWIN  B.  CRAGIN,  M.D.     Revised  by  FRANK  S.  MATHEWS, 
M.D. 

11.  ESSENTIALS  OF  DISEASES    OF  THE    SKIN.      6th  edition. 

61  illustrations.     By  H.  W.  STELWAGON,  M.D. 

12.  ESSENTIALS  OF    MINOR    SURGERY,  BANDAGING,  AND 

VENEREAL    DISEASES.      26.   ed.      78    illustrations.      By 
EDWARD  MARTIN,  M.D. 

13.  ESSENTIALS  OF  r.RNITO-URINARY  AND  VFNFRRAL_DIS- 


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21.  ESSENTIALS  OF  NERVOUS  DISEASES  AND  INSANITY. 
4th  ed.  53  illustrations.  By  JOHN  C.  SHAW,  M.  D.  Revised 
by  SMITH  ELY  JELLIFFE,  M.  D. 

24  ESSENTIALS  OF  DISEASES  OF  THE  EAR.  3^  ed.,  illus- 
trated. By  E.  BALDWIN  GLEASON,  M.  D. 

25.  ESSENTIALS  OF  HISTOLOGY.  3d  ed.  106  illustrations.  By 
Louis  LEROY,  M.  D. 

W.  B.  SAUNDERS  COMPANY,  925  w.inu«  &..  puu. 


